Optimized antibody compositions for treatment of ocular disorders

ABSTRACT

The present invention provides antibody conjugates that include antibodies (e.g., anti-VEGF antibodies) covalently linked to polymers (e.g., hyaluronic acid (HA) polymers), cysteine engineered antibodies, pharmaceutical compositions thereof, and uses thereof, for example for treatment of disorders associated with pathological angiogenesis (e.g., ocular disorders).

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 2, 2021, isnamed 50474-160004_Sequence_Listing_8_2_21_ST25 and is 57,729 bytes insize.

FIELD OF THE INVENTION

The invention relates generally to antibody conjugates, cysteineengineered antibodies, compositions (e.g., pharmaceutical compositions)thereof, and methods of use thereof.

BACKGROUND OF THE INVENTION

Angiogenesis is a tightly-regulated process through which new bloodvessels form from pre-existing blood vessels. Although angiogenesis isimportant during development to ensure adequate blood circulation, manydisorders are associated with pathological angiogenesis, such as oculardisorders (e.g., age-related macular degeneration, AMD) and cellproliferative disorders (e.g., cancer). Vascular endothelial growthfactor (VEGF) is a clinically-validated driver of angiogenesis, andneutralization of VEGF (e.g., using an anti-VEGF blocking antibody), canbe used to treat disorders associated with pathological angiogenesis.

Current approaches for treatment of ocular disorders associated withpathological angiogenesis (e.g., AMD (e.g., wet AMD), diabetic macularedema (DME), diabetic retinopathy (DR), and retinal vein occlusion(RVO)) typically involve intravitreal injection of VEGF antagonists(e.g., the anti-VEGF Fab ranibizumab). Because the site of action ofanti-VEGF Fabs is in the back of the eye at the retina, and also becauseFabs can have relatively short residence time in the eye, maximumpatient benefit from anti-VEGF Fabs is typically obtained by relativelyfrequent dosings (e.g., every four weeks, Q4W) by intravitrealinjection. Long-acting delivery of anti-VEGF antibodies or antibodyfragments (e.g., Fabs) for ocular disorders may be desired, at least inpart, to decrease dosing frequency, which can improve patientconvenience and compliance.

There remains a need for antibody compositions for long-acting deliveryfor treatment of ocular disorders (e.g., AMD (e.g., wet AMD), diabeticmacular edema (DME), diabetic retinopathy (DR), and retinal veinocclusion (RVO)).

SUMMARY OF THE INVENTION

The invention provides antibody conjugates that include monodispersepolymers (e.g., monodisperse hyaluronic acid (HA) polymers) covalentlylinked to antibodies (e.g., anti-VEGF antibodies), cysteine engineeredantibodies that can be used, e.g., in preparing antibody conjugates,compositions that include antibody conjugates (e.g., pharmaceuticalcompositions), as well as methods of making and using the same, forexample, for therapeutic uses.

In one aspect, the invention features an antibody conjugate comprising(i) an antibody and (ii) a hyaluronic acid (HA) polymer covalentlyattached to the antibody, wherein the HA polymer has a polydispersityindex (PDI) of 1.1 or lower. In some embodiments, the HA polymer has aPDI between 1.0 to 1.1. In some embodiments, the HA polymer has a PDIbetween 1.0 to about 1.05. In some embodiments, the HA polymer has a PDIbetween about 1.0001 to about 1.05. In some embodiments, the HA polymerhas a PDI of about 1.001. In some embodiments, the HA polymer has amolecular weight of about 1 megadalton (MDa) or lower. In someembodiments, the HA polymer has a molecular weight between about 25 kDaand about 500 kDa. In some embodiments, the HA polymer has a molecularweight between about 100 kDa and about 250 kDa. In some embodiments, theHA polymer has a molecular weight between about 150 kDa and about 200kDa. In some embodiments, the HA polymer is a linear HA polymer. In someembodiments, the antibody conjugate has a hydrodynamic radius betweenabout 10 nm and about 60 nm. In some embodiments, the antibody conjugatehas a hydrodynamic radius between about 25 nm and about 35 nm. In someembodiments, the hydrodynamic radius is about 20 nm to about 30 nm.

In some embodiments of the preceding aspect, the antibody conjugate hasan ocular half-life that is increased relative to a reference antibodythat is not covalently attached to the HA polymer. In some embodiments,the ocular half-life is increased at least about 2-fold relative to thereference antibody. In some embodiments, the ocular half-life isincreased at least about 4-fold relative to the reference antibody. Insome embodiments, the ocular half-life is a vitreal half-life. In someembodiments, the reference antibody is identical to the antibody of theantibody conjugate.

In some embodiments of the preceding aspect, the antibody specificallybinds to a biological molecule selected from the group consisting ofvascular endothelial growth factor (VEGF); IL-1β; IL-6; IL-6R; IL-13;IL-13R; PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3,αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin; complementfactor D; TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and a proteingenetically linked to AMD risk. In some embodiments, the VEGF receptoris VEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR. In some embodiments, theprotein genetically linked to AMD risk is selected from the groupconsisting of complement pathway components C2, factor B, factor H,CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1;TLR3; TLR4; CETP; LIPC, COL10A1; and TNFRSF10A.

In some embodiments of the preceding aspect, the antibody specificallybinds to VEGF. In some embodiments, the antibody comprises the followingsix hypervariable regions (HVRs): (a) an HVR-H1 comprising the aminoacid sequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising theamino acid sequence of GX₁TPX₂GGX₃X₄X₅YX₆DSVX₇X₈ (SEQ ID NO: 2), whereinX₁ is Ile or His, X₂ is Ala or Arg, X₃ is Tyr or Lys, X₄ is Thr or Glu,X₅ is Arg, Tyr, Gln, or Glu, X₆ is Ala or Glu, X₇ is Lys or Glu, and X₈is Gly or Glu; (c) an HVR-H3 comprising the amino acid sequence ofFVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of RASQX₁VSTAVA (SEQ ID NO: 4), wherein X₁ is Asp or Arg; (e)an HVR-L2 comprising the amino acid sequence of X₁ASFLYS (SEQ ID NO: 5),wherein X₁ is Ser or Met; and (f) an HVR-L3 comprising the amino acidsequence of X₁QGYGX2PFT (SEQ ID NO: 6), wherein X₁ is Gln, Asn, or Thrand X₂ is Ala, Asn, Gln, or Arg. In some embodiments, the antibodycomprises the following six HVRs: (a) an HVR-H1 comprising the aminoacid sequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising theamino acid sequence of GITPAGGYTRYADSVKG (SEQ ID NO: 7),GITPAGGYEYYADSVKG (SEQ ID NO: 21), or GITPAGGYEYYADSVEG (SEQ ID NO: 22);(c) an HVR-H3 comprising the amino acid sequence of FVFFLPYAMDY (SEQ IDNO: 3); (d) an HVR-L1 comprising the amino acid sequence of RASQDVSTAVA(SEQ ID NO: 8); (e) an HVR-L2 comprising the amino acid sequence ofSASFLYS (SEQ ID NO: 9); and (f) an HVR-L3 comprising the amino acidsequence of QQGYGAPFT (SEQ ID NO: 10) or QQGYGNPFT (SEQ ID NO: 23).

In some embodiments of the above aspect, the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some embodiments, the antibody further comprises the following heavychain variable (VH) domain framework regions (FRs): (a) an FR-H1comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTIS(SEQ ID NO: 13); (b) an FR-H2 comprising the amino acid sequence ofWVRQAPGKGLEWVA (SEQ ID NO: 14); (c) an FR-H3 comprising the amino acidsequence of RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) anFR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16).In some embodiments, the antibody further comprises the following lightchain variable (VL) domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20).

In some embodiments of the above aspect, the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGNPFT (SEQ ID NO: 23).In some embodiments, the antibody further comprises the following VLdomain FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 24); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20).

In some embodiments of the above aspect, the antibody comprises thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some embodiments, the antibody further comprises the following VLdomain FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ IDNO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18) orWYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19) orGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome embodiments, the antibody further comprises the following VH domainFRs: (a) an FR-H1 comprising the amino acid sequence ofEEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) an FR-H3comprising the amino acid sequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR(SEQ ID NO: 31); and (d) an FR-H4 comprising the amino acid sequence ofWGQGELVTVSS (SEQ ID NO: 32).

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising an amino acid sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 11, 40, or 42; (b) aVL domain comprising an amino acid sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 12, 41, or 46; or (c)a VH domain as in (a) and a VL domain as in (b). In some embodiments,the VH domain further comprises the following FRs: (a) an FR-H1comprising the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTIS(SEQ ID NO: 13); (b) an FR-H2 comprising the amino acid sequence ofWVRQAPGKGLEWVA (SEQ ID NO: 14) or WVRQEPGKGLEWVA (SEQ ID NO: 39); (c) anFR-H3 comprising the amino acid sequence ofRFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16). Insome embodiments, the VH domain comprises the amino acid sequence of SEQID NO: 11. In some embodiments, the VL domain further comprises thefollowing FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) or DIQMTQSPSSLSASVGDRVTIDC (SEQID NO: 45); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19),GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID NO: 44), orGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 54); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20) orFGQGTKVEVK (SEQ ID NO: 55). In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 12.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 11 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 40 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 42 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 42 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 41.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising an amino acid sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 33 or 51; (b) a VLdomain comprising an amino acid sequence having at least 95% sequenceidentity to the amino acid sequence of SEQ ID NO: 12, 34, 35, 36, 37, or38; or (c) a VH domain as in (a) and a VL domain as in (b). In someembodiments, the antibody further comprises the following FRs: (a) anFR-H1 comprising the amino acid sequence ofEEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) an FR-H3comprising the amino acid sequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR(SEQ ID NO: 31); and (d) an FR-H4 comprising the amino acid sequence ofWGQGELVTVSS (SEQ ID NO: 32). In some embodiments, the VH domaincomprises the amino acid sequence of SEQ ID NO: 33. In some embodiments,the VH domain comprises the amino acid sequence of SEQ ID NO: 51. Insome embodiments, the antibody further comprises the following FRs: (a)an FR-L1 comprising the amino acid sequence of DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25), orDIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18) orWYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19),GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24), orGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome embodiments, the VL domain comprises the amino acid sequence of SEQID NO: 34. In some embodiments, the VL domain comprises the amino acidsequence of SEQ ID NO: 35. In some embodiments, the VL domain comprisesthe amino acid sequence of SEQ ID NO: 36. In some embodiments, the VLdomain comprises the amino acid sequence of SEQ ID NO: 37. In someembodiments, the VL domain comprises the amino acid sequence of SEQ IDNO: 12. In some embodiments, the VL domain comprises the amino acidsequence of SEQ ID NO: 38.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 33 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 38.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 33 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 34.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 33 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 33 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 36.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 33 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 37.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 33 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 51 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 38.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 51 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 51 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 37.

In some embodiments of the above aspect, the antibody comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 51 and (b) a VLdomain comprising the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the above aspect, the antibody comprises (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 48 and (b)a light chain comprising the amino acid sequence of SEQ ID NO: 50.

In some embodiments of the above aspect, the antibody comprises (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b)a light chain comprising the amino acid sequence of SEQ ID NO: 50.

In some embodiments of any of the preceding aspects, the antibody iscapable of inhibiting the binding of VEGF to a VEGF receptor. In someembodiments, the VEGF receptor is VEGF receptor 1 (Flt-1). In someembodiments, the VEGF receptor is VEGF receptor 2 (KDR).

In some embodiments of any of the preceding aspects, the antibody bindshuman VEGF (hVEGF) with a Kd of about 2 nM or lower. In someembodiments, the antibody binds hVEGF with a Kd between about 75 pM andabout 2 nM. In some embodiments, the antibody binds hVEGF with a Kdbetween about 75 pM and about 600 pM. In some embodiments, the antibodybinds hVEGF with a Kd between about 75 pM and about 500 pM. In someembodiments, the antibody binds hVEGF with a Kd of about 80 pM. In someembodiments, the antibody binds hVEGF with a Kd of about 60 pM.

In some embodiments of any of the preceding aspects, the antibody has amelting temperature (Tm) of greater than about 83.5° C. In someembodiments, the antibody has a Tm of about 85° C. to about 91° C. Insome embodiments, the antibody has a Tm of about 89° C.

In some embodiments of any of the preceding aspects, the antibody has anisoelectric point (pI) of lower than 8. In some embodiments, theantibody has a pI from about 5 to about 7. In some embodiments, theantibody has a pI of from about 5 to about 6.

In some embodiments of any of the preceding aspects, the antibody ismonoclonal, human, humanized, or chimeric.

In some embodiments of any of the preceding aspects, the antibody is anantibody fragment that binds VEGF. In some embodiments, the antibodyfragment is selected from the group consisting of Fab, Fab-C, Fab′-SH,Fv, scFv, and (Fab′)₂ fragments. In some embodiments, the antibodyfragment is an Fab.

In some embodiments of any of the preceding aspects, the antibody is amonospecific antibody. In other embodiments of any of the precedingaspects, the antibody is a multispecific antibody. In some embodiments,the multispecific antibody is a bispecific antibody. In someembodiments, the bispecific antibody binds VEGF and a second biologicalmolecule selected from the group consisting of interleukin 1β (IL-1β);interleukin-6 (IL-6); interleukin-6 receptor (IL-6R); interleukin-13(IL-13); IL-13 receptor (IL-13R); PDGF; angiopoietin; angiopoietin 2;Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ;erythropoietin; complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2receptor; and a protein genetically linked to age-related maculardegeneration (AMD) risk. In some embodiments, the VEGF receptor isVEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF-receptor (mbVEGFR), orsoluble VEGF receptor (sVEGFR). In some embodiments, the proteingenetically linked to AMD risk is selected from the group consisting ofcomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1;TLR3; TLR4; CETP; LIPC, COL10A1; and TNFRSF10A.

In some embodiments of any of the preceding aspects, the antibody is acysteine engineered antibody. In some embodiments, the cysteineengineered antibody comprises a cysteine mutation in the heavy chainselected from the group consisting of HC-A118C, HC-A140C, and HC-L174C(EU numbering), or a cysteine mutation in the light chain selected fromthe group consisting of LC-K149C and LC-V205C (Kabat numbering). In someembodiments, the cysteine mutation in the heavy chain is HC-A1180 (EUnumbering). In some embodiments, the cysteine mutation in the heavychain is HC-A140C (EU numbering). In some embodiments, the cysteinemutation in the heavy chain is HC-L174C (EU numbering). In someembodiments, the cysteine mutation in the light chain is LC-K1490 (Kabatnumbering). In some embodiments, the cysteine mutation in the lightchain is LC-V205C (Kabat numbering). In some embodiments, the HA polymeris covalently attached to the antibody at the cysteine mutation.

In another aspect, any of the preceding antibody conjugates can be usedas a medicament.

In another aspect, any of the preceding antibody conjugates can be usedin the manufacture of a medicament for treating an ocular disorder in asubject.

In another aspect, any of the preceding antibody conjugates can be usedin reducing or inhibiting angiogenesis in a subject having an oculardisorder.

In another aspect, any of the preceding antibody conjugates can be usedin treating an ocular disorder in a subject.

In some embodiments of any of the preceding aspects, the ocular disorderis selected from the group consisting of age-related maculardegeneration (AMD), macular degeneration, macular edema, diabeticmacular edema (DME) (including focal, non-center DME and diffuse,center-involved DME), retinopathy, diabetic retinopathy (DR) (includingproliferative DR (PDR), non-proliferative DR (NPDR), and high-altitudeDR), other ischemia-related retinopathies, retinopathy of prematurity(ROP), retinal vein occlusion (RVO) (including central (CRVO) andbranched (BRVO) forms), CNV (including myopic CNV), cornealneovascularization, a disease associated with cornealneovascularization, retinal neovascularization, a disease associatedwith retinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, familial exudativevitreoretinopathy (FEVR), Coats' disease, Norrie Disease,Osteoporosis-Pseudoglioma Syndrome (OPPG), subconjunctival hemorrhage,rubeosis, ocular neovascular disease, neovascular glaucoma, retinitispigmentosa (RP), hypertensive retinopathy, retinal angiomatousproliferation, macular telangiectasia, iris neovascularization,intraocular neovascularization, retinal degeneration, cystoid macularedema (CME), vasculitis, papilloedema, retinitis, conjunctivitis(including infectious conjunctivitis and non-infectious (e.g., allergic)conjunctivitis), Leber congenital amaurosis, uveitis (includinginfectious and non-infectious uveitis), choroiditis, ocularhistoplasmosis, blepharitis, dry eye, traumatic eye injury, andSjögren's disease. In some embodiments, the ocular disorder is AMD, DME,DR, or RVO. In some embodiments, the ocular disorder is AMD. In someembodiments, the AMD is wet AMD. In some embodiments, the oculardisorder is DME.

In another aspect, the invention features a pharmaceutical compositioncomprising any of the antibody conjugates described herein apharmaceutically acceptable carrier, excipient, or diluent. In someembodiments, the pharmaceutical composition further comprises a secondagent, wherein the second agent is selected from the group consisting ofan antibody, an anti-angiogenic agent, a cytokine, a cytokineantagonist, a corticosteroid, an analgesic, and a compound that binds toa second biological molecule. In some embodiments, the anti-angiogenicagent is a VEGF antagonist. In some embodiments, the VEGF antagonist isan anti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGFreceptor fusion protein, an aptamer, an anti-VEGF DARPin®, or a VEGFRtyrosine kinase inhibitor. In some embodiments, the anti-VEGF antibodyis ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody.In some embodiments, the bispecific anti-VEGF antibody is ananti-VEGF/anti-Ang2 antibody. In some embodiments, theanti-VEGF/anti-Ang2 antibody is RG-7716. In some embodiments, thesoluble VEGF receptor fusion protein is aflibercept (EYLEA®). In someembodiments, the aptamer is pegaptanib (MACUGEN®). In some embodiments,the anti-VEGF DARPin® is abicipar pegol. In some embodiments, the VEGFRtyrosine kinase inhibitor is selected from the group consisting of4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT®(sunitinib). In some embodiments, the second biological molecule isselected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R;PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, andα5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D;TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and a protein geneticallylinked to AMD risk. In some embodiments, the VEGF receptor is VEGFR1,VEGFR2, VEGFR3, mbVEGFR, or sVEGFR. In some embodiments, the proteingenetically linked to AMD risk is selected from the group consisting ofcomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP;LIPC, COL10A1; and TNFRSF10A. In some embodiments, the compound thatbinds a second biological molecule is an antibody or antigen-bindingfragment thereof. In some embodiments, the antigen-binding antibodyfragment is selected from the group consisting of Fab, Fab-C, Fab′-SH,Fv, scFv, and (Fab′)₂ fragments. In some embodiments, theantigen-binding antibody fragment is an Fab.

In another aspect, any of the preceding pharmaceutical compositions canbe used as a medicament.

In another aspect, any of the preceding pharmaceutical compositions canbe used in the manufacture of a medicament for treating an oculardisorder in a subject.

In another aspect, any of the preceding pharmaceutical compositions canbe used in reducing or inhibiting angiogenesis in a subject having anocular disorder.

In another aspect, any of the preceding pharmaceutical compositions canbe used in treating an ocular disorder in a subject.

In some embodiments of any of the preceding aspects, the ocular disorderis selected from the group consisting of age-related maculardegeneration (AMD), macular degeneration, macular edema, diabeticmacular edema (DME) (including focal, non-center DME and diffuse,center-involved DME), retinopathy, diabetic retinopathy (DR) (includingproliferative DR (PDR), non-proliferative DR (NPDR), and high-altitudeDR), other ischemia-related retinopathies, retinopathy of prematurity(ROP), retinal vein occlusion (RVO) (including central (CRVO) andbranched (BRVO) forms), CNV (including myopic CNV), cornealneovascularization, a disease associated with cornealneovascularization, retinal neovascularization, a disease associatedwith retinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, familial exudativevitreoretinopathy (FEVR), Coats' disease, Norrie Disease,Osteoporosis-Pseudoglioma Syndrome (OPPG), subconjunctival hemorrhage,rubeosis, ocular neovascular disease, neovascular glaucoma, retinitispigmentosa (RP), hypertensive retinopathy, retinal angiomatousproliferation, macular telangiectasia, iris neovascularization,intraocular neovascularization, retinal degeneration, cystoid macularedema (CME), vasculitis, papilloedema, retinitis, conjunctivitis(including infectious conjunctivitis and non-infectious (e.g., allergic)conjunctivitis), Leber congenital amaurosis, uveitis (includinginfectious and non-infectious uveitis), choroiditis, ocularhistoplasmosis, blepharitis, dry eye, traumatic eye injury, andSjögren's disease. In some embodiments, the ocular disorder is AMD, DME,DR, or RVO. In some embodiments, the ocular disorder is AMD. In someembodiments, the AMD is wet AMD. In some embodiments, the oculardisorder is DME.

In another aspect, the invention features a method of reducing orinhibiting angiogenesis in a subject having an ocular disorder,comprising administering to the subject an effective amount of any ofthe antibody conjugates described herein or any of the pharmaceuticalcompositions described herein, thereby reducing or inhibitingangiogenesis in the subject.

In another aspect, the invention features a method of treating an oculardisorder, the method comprising administering an effective amount of anyof the antibody conjugates described herein or any of the pharmaceuticalcompositions described herein to a subject in need of such treatment.

In some embodiments of any of the preceding aspects, the ocular disorderis selected from the group consisting of age-related maculardegeneration (AMD), macular degeneration, macular edema, diabeticmacular edema (DME) (including focal, non-center DME and diffuse,center-involved DME), retinopathy, diabetic retinopathy (DR) (includingproliferative DR (PDR), non-proliferative DR (NPDR), and high-altitudeDR), other ischemia-related retinopathies, retinopathy of prematurity(ROP), retinal vein occlusion (RVO) (including central (CRVO) andbranched (BRVO) forms), CNV (including myopic CNV), cornealneovascularization, a disease associated with cornealneovascularization, retinal neovascularization, a disease associatedwith retinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, familial exudativevitreoretinopathy (FEVR), Coats' disease, Norrie Disease,Osteoporosis-Pseudoglioma Syndrome (OPPG), subconjunctival hemorrhage,rubeosis, ocular neovascular disease, neovascular glaucoma, retinitispigmentosa (RP), hypertensive retinopathy, retinal angiomatousproliferation, macular telangiectasia, iris neovascularization,intraocular neovascularization, retinal degeneration, cystoid macularedema (CME), vasculitis, papilloedema, retinitis, conjunctivitis(including infectious conjunctivitis and non-infectious (e.g., allergic)conjunctivitis), Leber congenital amaurosis, uveitis (includinginfectious and non-infectious uveitis), choroiditis, ocularhistoplasmosis, blepharitis, dry eye, traumatic eye injury, andSjögren's disease. In some embodiments, the ocular disorder is AMD, DME,DR, or RVO. In some embodiments, the ocular disorder is AMD. In someembodiments, the AMD is wet AMD. In some embodiments, the oculardisorder is DME.

In some embodiments of any of the preceding aspects, the method furthercomprises administering to the subject an effective amount of a secondagent, wherein the second agent is selected from the group consisting ofan antibody, an anti-angiogenic agent, a cytokine, a cytokineantagonist, a corticosteroid, an analgesic, and a compound that binds toa second biological molecule. In some embodiments, the anti-angiogenicagent is a VEGF antagonist. In some embodiments, the VEGF antagonist isan anti-VEGF antibody, an anti-VEGF receptor antibody, a soluble VEGFreceptor fusion protein, an aptamer, an anti-VEGF DARPin®, or a VEGFRtyrosine kinase inhibitor. In some embodiments, the anti-VEGF antibodyis ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody.In some embodiments, the bispecific anti-VEGF antibody is ananti-VEGF/anti-Ang2 antibody. In some embodiments, theanti-VEGF/anti-Ang2 antibody is RG-7716. In some embodiments, thesoluble VEGF receptor fusion protein is aflibercept (EYLEA®). In someembodiments, the aptamer is pegaptanib (MACUGEN®). In some embodiments,the anti-VEGF DARPin® is abicipar pegol. In some embodiments, the VEGFRtyrosine kinase inhibitor is selected from the group consisting of4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT®(sunitinib). In some embodiments, the second biological molecule isselected from the group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R;PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, andα5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D;TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and a protein geneticallylinked to AMD risk. In some embodiments, the VEGF receptor is VEGFR1,VEGFR2, VEGFR3, mbVEGFR, or sVEGFR. In some embodiments, the proteingenetically linked to AMD risk is selected from the group consisting ofcomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP;LIPC, COL10A1; and TNFRSF10A. In some embodiments, the compound thatbinds a second biological molecule is an antibody or antigen-bindingfragment thereof. In some embodiments, the antigen-binding antibodyfragment is selected from the group consisting of Fab, Fab-C, Fab′-SH,Fv, scFv, and (Fab′)₂ fragments.

In some embodiments of any of the preceding aspects, the antibodyconjugate or the pharmaceutical composition is administeredintravitreally, ocularly, intraocularly, juxtasclerally, subtenonly,superchoroidally, topically, intravenously, intramuscularly,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intrathecally, intranasally,intravaginally, intrarectally, topically, intratumorally,intraperitoneally, peritoneally, intraventricularly, subcutaneously,subconjunctivally, intravesicularly, mucosally, intrapericardially,intraumbilically, intraorbitally, orally, transdermally, by inhalation,by injection, by eye drop, by implantation, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions. In someembodiments, the antibody conjugate or the pharmaceutical composition isadministered intravitreally, ocularly, intraocularly, juxtasclerally,subtenonly, superchoroidally, or topically. In some embodiments, theantibody conjugate or the pharmaceutical composition is administeredintravitreally by injection. In some embodiments, the antibody conjugateor the pharmaceutical composition is administered topically by eye dropor ointment. In some embodiments, the antibody conjugate or thepharmaceutical composition is administered by a port delivery device.

In some embodiments of any of the preceding aspects, the subject is ahuman.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent or patent application with color drawings will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1A is a graph showing a population distribution of molecularweights (in terms of molar mass) in a representative sample of 200 kDaHA.

FIG. 1B is a graph showing a population distribution of the number ofmaleimides on a 200 kDa HA chain resulting from a Monte Carlo simulationof stochastic modification with each acid group having a 5% chance ofmodification.

FIG. 1C is a graph showing the polydispersity of 40 kDa, 200 kDa, and600 kDa HA polymers. The table below the graph shows the number-averagemolecular weight (Mn), weight-average molecular weight (Mw),polydispersity index (PDI), and molecular weight (MW) range (in terms ofMw) for the indicated samples.

FIG. 2 is a series of graphs showing that HA-G6.31.AARR conjugates havedifferences in physical stability under physiologically relevant stressconditions as assessed by size exclusion chromatography (SEC) in-linewith refractive index (RI) and multi-angle light scattering (MALS)detectors (SEC-RI-MALS). The series labels refer to the HA backbonemolecular weight (40 kDa (“40K”); 200 kDa (“200K”); and 600 kDa (“600K”)and Fab loading level.

FIG. 3 is a series of graphs showing changes in SEC retention profilesover time for HA40K-G6.31.AARR-4.7% (left panel), HA200K-G6.31.AARR-4.7%(center panel), and HA600K-G6.31.AARR-2.1% (right panel), demonstratingthat SEC retention times shift to later times (smaller hydrodynamicsize), with the extent of this shift dependent on HA backbone molecularweight.

FIG. 4 is a series of graphs showing SEC-RI-MALS data plotted ascumulative weight fraction for HA40K-G6.31.AARR-4.7% (left panel),HA200K-G6.31.AARR-4.7% (center panel), and HA600K-G6.31.AARR-2.1% (rightpanel).

FIG. 5 is a graph showing the results of SEC-RI-MALS characterization ofcommercial polydisperse HA (black) and monodisperse HA (grey),demonstrating the difference in mass distributions between the twoproduction techniques. The table in the right panel shows the Mn, Mw,and PDI values determined by this analysis.

FIG. 6 is a graph showing the results of SEC-RI-MALS characterization ofpolydisperse HA200K-G6.31.AARR (black) and monodisperseHA150K-G6.31.AARR (grey), demonstrating the difference in massdistributions. The table in the right panel shows the Mn, Mw, and PDIvalues determined by this analysis.

FIG. 7 is a schematic diagram showing that standard Fab-C formatmolecules are designed to contain a free cysteine residue useful forconjugation by extending the standard Fab hinge peptide sequence to thefirst or second hinge disulfide cysteine. To minimize or preventscrambling between the interchain disulfide and this free cysteineresidue, the cysteine can instead be mutated onto the surface of the Fabat a location spatially further separated from the interchain disulfide(referred to herein as “ThioFabs”).

FIGS. 8A-8C are a series of schematic diagrams showing that theflexibility and spatial proximity of the hinge sequence can lead torearrangement into three possible disulfide states leaving threedifferent cysteine residues available for conjugation. FIG. 8A shows theintended configuration in which the hinge sequence cysteine is reducedand available for conjugation. FIG. 8B shows a cyclized heavy chain (HC)variant in which the hinge cysteine forms a disulfide bond with the HCcysteine residue that normally is part of the interchain disulfide bond,leaving the light chain (LC) interchain disulfide cysteine available forconjugation. FIG. 8C shows a LC variant in which the hinge cysteineforms a disulfide bond with the LC cysteine that normally forms part ofthe interchain disulfide bond, leaving the HC interchain disulfidecysteine available for conjugation.

FIG. 9 shows a series of graphs showing the results of a series ofmaleimide capping and limited Lys-C digestion experiments performed onG6.31.AARR.Fab-C and analyzed by reverse phase ultra performance liquidchromatography time-of-flight (RP-UPLC-TOF) mass spectrometry. Thenumbers over the peaks are total ion counts for that peak and are ameasure of peak area.

FIGS. 10A and 10B are a series of graphs showing the results of limitedLys-C (FIG. 10A) and hyaluronidase (HAase) (FIG. 10B) enzymatic digestsof HA-G6.31.AARR conjugates, confirming the presence of conjugationvariants associated with conjugation through both cysteines normallyoccupied by the interchain disulfide.

FIG. 11 is a series of graphs showing that conjugation of G6.31.AARRThioFabs to HA200K-maleimide proceeded normally compared toG6.31.AARR.Fab-C, although the conversion of Fab to conjugate was lowerfor the ThioFab samples.

FIG. 12 is a graph showing deconjugation of a model polyethylene glycol(PEG)-maleimide polymer from G6.31.AARR of different formats in PBS+2 mMoxidized glutathione (GSSG) at 37° C. as assessed by RP-UPLC-TOF.

FIG. 13A shows the Kabat numbering scheme for the 4D5 light chain.

FIG. 13B shows a sequential numbering scheme (left column) starting atthe N-terminus in comparison with the Kabat numbering scheme (middlecolumn) and EU numbering scheme (right column) for the 4D5 antibody.

FIG. 14 is a graph showing that HA-G6.31.AARR conjugates produced frommonodisperse HA show improved physical stability under physiologicalstress at four weeks compared to HA-G6.31.AARR conjugates of similarsize produced from polydisperse HA. The table in the right panel showsMw (kDa) at weeks 0, 2, and 4.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs) and/or frameworkregions (FRs), compared to a parent antibody which does not possess suchalterations, such alterations resulting in an improvement in theaffinity of the antibody for antigen.

The term “vascular endothelial growth factor” or “VEGF” refers tovascular endothelial growth factor protein A, as exemplified by SEQ IDNO: 47 (see also Swiss Prot Accession Number P15692, Gene ID (NCBI):7422). The term “VEGF” encompasses the protein having the amino acidsequence of SEQ ID NO: 47 as well as homologues and isoforms thereof.The term “VEGF” also encompasses the known isoforms, e.g., spliceisoforms, of VEGF, e.g., VEGF₁₁₁, VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉,and VEGF₂₀₆, together with the naturally-occurring allelic and processedforms thereof, including the 110-amino acid human vascular endothelialcell growth factor generated by plasmin cleavage of VEGF₁₆₅ as describedin Ferrara Mol. Biol. Cell. 21:687 (2010), Leung et al., Science,246:1306 (1989), and Houck et al., Mol. Endocrin., 5:1806 (1991). Theterm “VEGF” also refers to VEGFs from non-human species such as mouse,rat or primate. Sometimes the VEGF from a specific species are indicatedby terms such as hVEGF for human VEGF, mVEGF for murine VEGF, and thelike. The term “VEGF” is also used to refer to truncated forms of thepolypeptide comprising amino acids 8 to 109 or 1 to 109 of the 165-aminoacid human vascular endothelial cell growth factor. Reference to anysuch forms of VEGF may be identified in the present application, e.g.,by “VEGF₁₀₉,” “VEGF (8-109),” “VEGF (1-109)” or “VEGF₁₆₅.” The aminoacid positions for a “truncated” native VEGF are numbered as indicatedin the native VEGF sequence. For example, amino acid position 17(methionine) in truncated native VEGF is also position 17 (methionine)in native VEGF. The truncated native VEGF has binding affinity for theKDR and Flt-1 receptors comparable to native VEGF. The term “VEGFvariant” as used herein refers to a VEGF polypeptide which includes oneor more amino acid mutations in the native VEGF sequence. Optionally,the one or more amino acid mutations include amino acid substitution(s).For purposes of shorthand designation of VEGF variants described herein,it is noted that numbers refer to the amino acid residue position alongthe amino acid sequence of the putative native VEGF (provided in Leunget al., supra and Houck et al., supra). Unless specified otherwise, theterm “VEGF” as used herein indicates VEGF-A.

The terms “anti-VEGF antibody,” an “antibody that binds to VEGF,” and“antibody that specifically binds VEGF” refer to an antibody that iscapable of binding VEGF with sufficient affinity such that the antibodyis useful as a diagnostic and/or therapeutic agent in targeting VEGF. Inone embodiment, the extent of binding of an anti-VEGF antibody to anunrelated, non-VEGF protein is less than about 10% of the binding of theantibody to VEGF as measured, for example, by a radioimmunoassay (RIA).In certain embodiments, an antibody that binds to VEGF has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g., from 10⁻⁸ M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M). In certain embodiments, an anti-VEGFantibody binds to an epitope of VEGF that is conserved among VEGF fromdifferent species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab-C, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); and multispecific antibodies formed from antibodyfragments. In some instances, examples of antibody fragments include butare not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linearantibodies; single-chain antibody molecules (e.g., scFv); andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire light (L) chain along with the variableregion domain of the heavy (H) chain (VH), and the first constant domainof one heavy chain (CH1). Pepsin treatment of an antibody yields asingle large F(ab′)₂ fragment which roughly corresponds to two disulfidelinked Fab fragments having divalent antigen-binding activity and isstill capable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having additional few residues at the carboxy terminus ofthe CH1 domain including one or more cysteines from the antibody hingeregion. Fab-C molecules are Fab molecules that are expressed such thatthe sequence is truncated at the first hinge cysteine, resulting in aFab with a free cysteine directly upon expression (see, e.g., Shatz etal. Mol. Pharmaceutics 2016; PubMed identifier (PMID) 27244474). Forexample, a Fab-C molecule may have a free cysteine at position Cys227 ofthe heavy chain. In other instances, a Fab-C molecule may have a freecysteine at position Cys229 of the heavy chain. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991).

“Fv” consists of a dimer of one heavy- and one light-chain variableregion domain in tight, non-covalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeHVRs specific for an antigen) has the ability to recognize and bindantigen, although often at a lower affinity than the entire bindingsite.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the VH and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the VH and VL domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the VH and VL domains of the twoantibodies are present on different polypeptide chains. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

A “cysteine engineered antibody” or “cysteine engineered antibodyvariant” is an antibody in which one or more residues of an antibody aresubstituted with cysteine residues. In certain instances, cysteineengineered antibodies may be referred to as THIOMAB™ antibodies orThioFab antibodies. The thiol group(s) of the cysteine engineeredantibodies can be conjugated to other moieties, e.g., polymers (e.g., HApolymers, including monodisperse HA polymers). In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such aspolymers (e.g., HA polymers). For example, a cysteine engineeredantibody may be an antibody with a single mutation of a non-cysteinenative residue to a cysteine in the light chain (e.g., LC-G64C,LC-I106C, LC-R108C, LC-R142C, or LC-K149C according to Kabat numbering)or in the heavy chain (e.g., HC-D101C, HC-V184C, or HC-T205C accordingto Kabat numbering, or HC-T114C, HC-A140C, HC-L174C, HC-L179C, HC-T187C,HC-T209C, HC-V262C, HC-G371C, HC-Y373C, HC-E382C, HC-S424C, HC-N434C,and HC-Q438C according to EU numbering (i.e., HC-A136C according toKabat numbering is HC-A140C according to EU numbering)) (see FIGS. 13Aand 13B). In particular instances, a cysteine engineered antibody mayinclude a cysteine mutation in the heavy chain selected from the groupconsisting of HC-A118C, HC-A140C, and HC-L174C (EU numbering), or acysteine mutation in the light chain selected from the group consistingof LC-V205C and LC-K149C (Kabat numbering). In some instances, acysteine engineered antibody has a single cysteine mutation in eitherthe heavy or light chain such that each full-length antibody (i.e., anantibody with two heavy chains and two light chains) has two engineeredcysteine residues, and each Fab fragment has one engineered cysteineresidue. In other instances, a cysteine engineered antibody has morethan one cysteine mutations (e.g., 2, 3, 4, or 5 cysteine mutations).

A “free cysteine amino acid” refers to a cysteine amino acid residuewhich has been engineered into a parent antibody, has a thiol functionalgroup (—SH), and is not paired as an intramolecular or intermoleculardisulfide bridge.

The term “thiol reactivity value” is a quantitative characterization ofthe reactivity of free cysteine amino acids. The thiol reactivity valueis the percentage of a free cysteine amino acid in a cysteine engineeredantibody which reacts with a thiol-reactive reagent, and converted to amaximum value of 1. For example, a free cysteine amino acid on acysteine engineered antibody which reacts in 100% yield with athiol-reactive reagent, such as a biotin-maleimide reagent, to form abiotin-labelled antibody has a thiol reactivity value of 1.0. Anothercysteine amino acid engineered into the same or different parentantibody which reacts in 90% yield with a thiol-reactive reagent has athiol reactivity value of about 0.9. Another cysteine amino acidengineered into the same or different parent antibody which reacts in80% yield with a thiol-reactive reagent has a thiol reactivity value ofabout 0.8. Another cysteine amino acid engineered into the same ordifferent parent antibody which reacts in 70% yield with athiol-reactive reagent has a thiol reactivity value of about 0.7.Another cysteine amino acid engineered into the same or different parentantibody which reacts in 60% yield with a thiol-reactive reagent has athiol reactivity value of about 0.6. Another cysteine amino acidengineered into the same or different parent antibody which reacts in50% yield with a thiol-reactive reagent has a thiol reactivity value ofabout 0.5. Another cysteine amino acid engineered into the same ordifferent parent antibody which reacts in 40% yield with athiol-reactive reagent has a thiol reactivity value of about 0.4.Another cysteine amino acid engineered into the same or different parentantibody which reacts in 30% yield with a thiol-reactive reagent has athiol reactivity value of about 0.3. Another cysteine amino acidengineered into the same or different parent antibody which reacts in20% yield with a thiol-reactive reagent has a thiol reactivity value ofabout 0.2. Another cysteine amino acid engineered into the same ordifferent parent antibody which reacts in 10% yield with athiol-reactive reagent has a thiol reactivity value of about 0.1.Another cysteine amino acid engineered into the same or different parentantibody which fails totally to react with a thiol-reactive reagent hasa thiol reactivity value of 0. Determination of the thiol reactivityvalue of a particular cysteine may be conducted by ELISA assay (e.g., aPHESELECTOR assay as described herein), mass spectroscopy, liquidchromatography, autoradiography, or other quantitative analytical tests.

A “parent antibody” is an antibody comprising an amino acid sequencefrom which one or more amino acid residues are replaced by one or morecysteine residues. The parent antibody may comprise a native or wildtype sequence. The parent antibody may have pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions) relative to other native, wild type, or modified forms ofan antibody. A parent antibody may be directed against a target antigenof interest, e.g., a biologically important polypeptide, such as VEGF.Any of the antibodies described herein (e.g., anti-VEGF antibodies) maybe a parent antibody.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor); and Bcell activation.

“Framework” or “framework region” or “FR” refers to variable domainresidues other than hypervariable region (HVR) residues. The FR of avariable domain generally consists of four FR domains: FR1, FR2, FR3,and FR4.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, FR residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies can comprise residues thatare not found in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. Thevariable or “V” domain mediates antigen binding and defines specificityof a particular antibody for its particular antigen. However, thevariability is not evenly distributed across the span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The term “hypervariable region” or“HVR” when used herein refers to the amino acid residues of an antibodywhich are responsible for antigen-binding. The hypervariable regiongenerally comprises amino acid residues from, for example, around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and aroundabout residues 26-35 (H1), 49-65 (H2) and 95-102 (H3) in the VH (in oneembodiment, H1 is around about residues 31-35); Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (e.g., residues 26-32 (L1), 50-52(L2), and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2), and 96-101(H3) in the VH; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987). Thevariable domains of native heavy and light chains each comprise fourFRs, largely adopting a beta-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies (see Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Accordingly, theHVR and FR sequences generally appear in the following sequence in VH(or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity (ADCC).

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (see, e.g., Kabat et al.,Sequences of Immunological Interest. 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), which is hereinincorporated by reference in its entirety). The “EU numbering system” or“EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system,also called the EU index, as described in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md., 1991.

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

The term an “isolated antibody” when used to describe the variousantibodies disclosed herein, means an antibody that has been identifiedand separated and/or recovered from a cell or cell culture from which itwas expressed. Contaminant components of its natural environment arematerials that would typically interfere with diagnostic or therapeuticuses for the polypeptide, and can include enzymes, hormones, and otherproteinaceous or non-proteinaceous solutes. In some embodiments, anantibody is purified to greater than 95% or 99% purity as determined by,for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing(IEF), capillary electrophoresis) or chromatographic (e.g., ion exchangeor reverse phase HPLC). For a review of methods for assessment ofantibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007). In preferred embodiments, the antibody will bepurified (1) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (2) to homogeneity by SDS-PAGE under non-reducing orreducing conditions using Coomassie blue or, preferably, silver stain.Isolated antibody includes antibodies in situ within recombinant cells,because at least one component of the polypeptide natural environmentwill not be present. Ordinarily, however, isolated polypeptide will beprepared by at least one purification step.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “multispecific antibody” is used in the broadest sense andspecifically covers an antibody comprising a heavy chain variable domain(VH) and a light chain variable domain (VL), where the VH-VL unit haspolyepitopic specificity (i.e., is capable of binding to two differentepitopes on one biological molecule or each epitope on a differentbiological molecule). Such multispecific antibodies include, but are notlimited to, full-length antibodies, antibodies having two or more VL andVH domains, antibody fragments such as Fab, Fab′, Fab-C. Fv, dsFv, scFv,diabodies, bispecific diabodies and triabodies, antibody fragments thathave been linked covalently or non-covalently. “Polyepitopicspecificity” refers to the ability to specifically bind to two or moredifferent epitopes on the same or different target(s). “Dualspecificity” or “bispecificity” refers to the ability to specificallybind to two different epitopes on the same or different target(s).However, in contrast to bispecific antibodies, dual-specific antibodieshave two antigen-binding arms that are identical in amino acid sequenceand each Fab arm is capable of recognizing two antigens.Dual-specificity allows the antibodies to interact with high affinitywith two different antigens as a single Fab or IgG molecule. Accordingto one embodiment, the multispecific antibody in an IgG1 form binds toeach epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1μM to 0.001 pM, 0.5 μM to 0.001 pM or 0.1 μM to 0.001 pM. “Monospecific”refers to the ability to bind only one epitope.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

With regard to the binding of a antibody to a target molecule, the term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetmeans binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule. For example, specific binding can be determined by competitionwith a control molecule that is similar to the target, for example, anexcess of non-labeled target. In this case, specific binding isindicated if the binding of the labeled target to a probe iscompetitively inhibited by excess unlabeled target. The term “specificbinding” or “specifically binds to” or is “specific for” a particularpolypeptide or an epitope on a particular polypeptide target as usedherein can be exhibited, for example, by a molecule having a Kd for thetarget of 10⁻⁴M or lower, alternatively 10⁻⁵M or lower, alternatively10⁻⁶ M or lower, alternatively 10⁻⁷ M or lower, alternatively 10⁻⁸ M orlower, alternatively 10⁻⁹ M or lower, alternatively 10⁻¹⁰ M or lower,alternatively 10⁻¹¹ M or lower, alternatively 10⁻¹² M or lower or a Kdin the range of 10⁻⁴ M to 10⁻⁶ M or 10⁻⁶ M to 10⁻¹⁰ M or 10⁻⁷ M to 10⁻⁹M. As will be appreciated by the skilled artisan, affinity and Kd valuesare inversely related. A high affinity for an antigen is measured by alow Kd value. In one embodiment, the term “specific binding” refers tobinding where a molecule binds to a particular polypeptide or epitope ona particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope.

A “nucleic acid encoding an anti-VEGF antibody” refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y, where X is the number of amino acid residues scored asidentical matches by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. Unless specifically statedotherwise, all % amino acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

As used herein, “administering” is meant a method of giving a dosage ofa compound (e.g., an antibody (e.g., a cysteine engineered anti-VEGFantibody) or an antibody conjugate (e.g., a monodisperse HA conjugate)of the invention) or a composition (e.g., a pharmaceutical composition,e.g., a pharmaceutical composition including an antibody or an antibodyconjugate of the invention) to a subject. The compositions utilized inthe methods described herein can be administered, for example,intravitreally (e.g., by intravitreal injection), by eye drop,intramuscularly, intravenously, intradermally, percutaneously,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intrathecally, intranasally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctivally,intravesicularly, mucosally, intrapericardially, intraumbilically,intraocularly, intraorbitally, orally, topically, transdermally, byinhalation, by injection, by implantation, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions. Thecompositions utilized in the methods described herein can also beadministered systemically or locally. The method of administration canvary depending on various factors (e.g., the compound or compositionbeing administered and the severity of the condition, disease, ordisorder being treated).

“Angiogenesis” refers to the process through which new blood vesselsform from pre-existing blood vessels. Angiogenesis is distinct fromvasculogenesis, which is the de novo formation of endothelial cells frommesoderm cell precursors. Disorders associated with pathologicalangiogenesis can be treated by compositions and methods of theinvention. Exemplary disorders associated with pathological angiogenesisinclude but are not limited to ocular conditions (non-limiting ocularconditions include, for example, retinopathy including proliferativediabetic retinopathy, choroidal neovascularization (CNV), age-relatedmacular degeneration (AMD), diabetic and other ischemia-relatedretinopathies, diabetic macular edema (DME), pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, retinal vein occlusion(including central (CRVO) and branched (BRVO) forms), cornealneovascularization, retinal neovascularization, retinopathy ofprematurity (ROP), familial exudative vitreoretinopathy (FEVR), Coats'disease, Norrie Disease, Osteoporosis-Pseudoglioma Syndrome (OPPG),subconjunctival hemorrhage, rubeosis, ocular neovascular disease,neovascular glaucoma, and hypertensive retinopathy). Additional oculardisorders are described below.

The term “ocular disorder,” as used herein, includes any ocular disorder(also referred to interchangeably herein as “ocular condition”)associated with pathogical angiogenesis. An ocular disorder may becharacterized by altered or unregulated proliferation and/or invasion ofnew blood vessels into the structures of ocular tissues such as theretina or cornea. Non-limiting ocular disorders include, for example,AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, andgeographic atrophy (GA)), macular degeneration, macular edema, DME(e.g., focal, non-center DME and diffuse, center-involved DME),retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR (PDR),non-proliferative DR (NPDR), and high-altitude DR), otherischemia-related retinopathies, ROP, retinal vein occlusion (RVO) (e.g.,central (CRVO) and branched (BRVO) forms), CNV (e.g., myopic CNV),corneal neovascularization, diseases associated with cornealneovascularization, retinal neovascularization, diseases associated withretinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats' disease,Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis, ocularneovascular disease, neovascular glaucoma, retinitis pigmentosa (RP),hypertensive retinopathy, retinal angiomatous proliferation, maculartelangiectasia, iris neovascularization, intraocular neovascularization,retinal degeneration, cystoid macular edema (CME), vasculitis,papilloedema, retinitis, conjunctivitis (e.g., infectious conjunctivitisand non-infectious (e.g., allergic) conjunctivitis), Leber congenitalamaurosis (also known as Leber's congenital amaurosis or LCA), uveitis(including infectious and non-infectious uveitis), choroiditis (e.g.,multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye,traumatic eye injury, Sjögren's disease, and other ophthalmic diseaseswherein the disease or disorder is associated with ocularneovascularization, vascular leakage, and/or retinal edema. Additionalexemplary ocular disorders include diseases associated with rubeosis(neovascularization of the angle) and diseases caused by the abnormalproliferation of fibrovascular or fibrous tissue, including all forms ofproliferative vitreoretinopathy.

Exemplary diseases associated with corneal neovascularization include,but are not limited to, epidemic keratoconjunctivitis, vitamin Adeficiency, contact lens overwear, atopic keratitis, superior limbickeratitis, terygium keratitis sicca, Sjögren's syndrome, acne rosacea,phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration,chemical burns, bacterial ulcers, fungal ulcers, Herpes simplexinfections, Herpes zoster infections, protozoan infections, Kaposisarcoma, Mooren ulcer, Terrien's marginal degeneration, marginalkeratolysis, rheumatoid arthritis, systemic lupus, polyarteritis,trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome,periphigoid radial keratotomy, and corneal graph rejection.

Exemplary diseases associated with retinal/choroidal neovascularizationinclude, but are not limited to, diabetic retinopathy, maculardegeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthomaelasticum, Paget's disease, vein occlusion, artery occlusion, carotidobstructive disease, chronic uveitis/vitritis, mycobacterial infections,Lyme's disease, systemic lupus erythematosis, retinopathy ofprematurity, retinitis pigmentosa, retina edema (including macularedema), Eales disease, Behcet's disease, infections causing retinitis orchoroiditis (e.g., multifocal choroidits), presumed ocularhistoplasmosis, Best's disease (vitelliform macular degeneration),myopia, optic pits, Stargart's disease, pars planitis, retinaldetachment (e.g., chronic retinal detachment), hyperviscosity syndromes,toxoplasmosis, trauma, and post-laser complications.

An “angiogenic factor or agent” is a growth factor which stimulates thedevelopment of blood vessels, e.g., promote angiogenesis, endothelialcell growth, stability of blood vessels, and/or vasculogenesis, etc. Forexample, angiogenic factors, include, but are not limited to, e.g., VEGFand members of the VEGF family, PIGF, PDGF family, fibroblast growthfactor family (FGFs), TIE ligands (Angiopoietins), ephrins, Del-1,fibroblast growth factors: acidic (aFGF) and basic (bFGF), Follistatin,Granulocyte colony-stimulating factor (G-CSF), Hepatocyte growth factor(HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine,Placental growth factor, Platelet-derived endothelial cell growth factor(PD-ECGF), Platelet-derived growth factor, especially PDGF-BB orPDGFR-beta, Pleiotrophin (PTN), Progranulin, Proliferin, Transforminggrowth factor-alpha (TGF-alpha), Transforming growth factor-beta(TGF-beta), Tumor necrosis factor-alpha (TNF-alpha), Vascularendothelial growth factor (VEGF)/vascular permeability factor (VPF),etc. It would also include factors that accelerate wound healing, suchas growth hormone, insulin-like growth factor-I (IGF-I), VIGF, epidermalgrowth factor (EGF), CTGF and members of its family, and TGF-alpha andTGF-beta. See, for example, Klagsbrun and D'Amore, Annu. Rev. Physiol,53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003);Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini etal., Oncogene, 22:6549-6556 (2003) (e.g., Table 1 listing knownangiogenic factors); and Sato, Int. J. Clin. Oncol, 8:200-206 (2003).

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Itshould be understood that the anti-angiogenesis agent includes thoseagents that bind and block the angiogenic activity of the angiogenicfactor or its receptor. For example, an anti-angiogenesis agent is anantibody or other antagonist to an angiogenic agent as defined above,e.g., VEGF antagonists (e.g., antibodies to VEGF-A or to the VEGF-Areceptor (e.g., KDR receptor or Flt-1 receptor)), PDGF antagonists(e.g., anti-PDGFR inhibitors such as GLEEVEC™ (Imatinib Mesylate)).Anti-angiogenesis agents also include native angiogenesis inhibitors,e.g., angiostatin, endostatin, etc. See, for example, Klagsbrun andD'Amore, Annu. Rev. Physiol, 53:217-39 (1991); Streit and Detmar,Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenictherapy in malignant melanoma); Ferrara & Alitalo, Nature Medicine5(12):1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003)(e.g., Table 2 listing known antiangiogenic factors); and, Sato Int. J.Clin. Oncol, 8:200-206 (2003) (e.g., Table 1 lists anti-angiogenicagents used in clinical trials).

The term “VEGF antagonist,” as used herein, refers to a molecule capableof binding to VEGF, reducing VEGF expression levels, or neutralizing,blocking, inhibiting, abrogating, reducing, or interfering with VEGFbiological activities, including, but not limited to, VEGF binding toone or more VEGF receptors, VEGF signaling, and VEGF-mediatedangiogenesis and endothelial cell survival or proliferation. Forexample, a molecule capable of neutralizing, blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities canexert its effects by binding to one or more VEGF receptor (VEGFR) (e.g.,VEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF receptor (mbVEGFR), orsoluble VEGF receptor (sVEGFR)). Included as VEGF antagonists useful inthe methods of the invention are polypeptides that specifically bind toVEGF, anti-VEGF antibodies and antigen-binding fragments thereof,receptor molecules and derivatives which bind specifically to VEGFthereby sequestering its binding to one or more receptors, fusionsproteins (e.g., VEGF-Trap (Regeneron)), and VEGF₁₂₁-gelonin (Peregrine).VEGF antagonists also include antagonist variants of VEGF polypeptides,antisense nucleobase oligomers complementary to at least a fragment of anucleic acid molecule encoding a VEGF polypeptide; small RNAscomplementary to at least a fragment of a nucleic acid molecule encodinga VEGF polypeptide; ribozymes that target VEGF; peptibodies to VEGF; andVEGF aptamers. VEGF antagonists also include polypeptides that bind toVEGFR, anti-VEGFR antibodies, and antigen-binding fragments thereof, andderivatives which bind to VEGFR thereby blocking, inhibiting,abrogating, reducing, or interfering with VEGF biological activities(e.g., VEGF signaling), or fusions proteins. VEGF antagonists alsoinclude nonpeptide small molecules that bind to VEGF or VEGFR and arecapable of blocking, inhibiting, abrogating, reducing, or interferingwith VEGF biological activities. Thus, the term “VEGF activities”specifically includes VEGF-mediated biological activities of VEGF. Incertain embodiments, the VEGF antagonist reduces or inhibits, by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, theexpression level or biological activity of VEGF. In some embodiments,the VEGF inhibited by the VEGF-specific antagonist is VEGF (8-109), VEGF(1-109), or VEGF₁₆₅.

As used herein VEGF antagonists can include, but are not limited to,anti-VEGFR2 antibodies and related molecules (e.g., ramucirumab,tanibirumab, aflibercept), anti-VEGFR1 antibodies and related molecules(e.g., icrucumab, aflibercept (VEGF Trap-Eye; EYLEA®), andziv-aflibercept (VEGF Trap; ZALTRAP®)), bispecific VEGF antibodies(e.g., MP-0250, vanucizumab (VEGF-ANG2), and bispecific antibodiesdisclosed in US 2001/0236388), bispecific antibodies includingcombinations of two of anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms,anti-VEGF antibodies (e.g., bevacizumab, sevacizumab, and ranibizumab),and nonpeptide small molecule VEGF antagonists (e.g., pazopanib,axitinib, vandetanib, stivarga, cabozantinib, lenvatinib, nintedanib,orantinib, telatinib, dovitinig, cediranib, motesanib, sulfatinib,apatinib, foretinib, famitinib, and tivozanib). Additional VEGFantagonists are described below.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human. A “subject” may be a “patient.”

A “disorder” is any condition that would benefit from treatment with theantibody. For example, mammals who suffer from or need prophylaxisagainst abnormal angiogenesis (excessive, inappropriate or uncontrolledangiogenesis). This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question. Non-limiting examples of disorders to betreated herein include disorders associated with pathologicalangiogenesis (e.g., ocular disorders).

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient (e.g., an antibody conjugate) contained therein to beeffective, and which contains no additional components which areunacceptably toxic to a subject to which the formulation would beadministered.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, decreasing the rate of disease progression,amelioration or palliation of the disease state, and remission orimproved prognosis. In some embodiments, antibody conjugates of theinvention or other compositions that include an antibody conjugate ofthe invention (e.g., a pharmaceutical formulation) are used to delaydevelopment of a disease or to slow the progression of a disease.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordinarilyexpress the antibody where, for example, the nucleic acid molecule is ina chromosomal location different from that of natural cells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

A “variant” or “mutant” of a starting or reference polypeptide (e.g., areference antibody or its variable domain(s)/HVR(s)), is a polypeptidethat (1) has an amino acid sequence different from that of the startingor reference polypeptide and (2) was derived from the starting orreference polypeptide through either natural or artificial (man-made)mutagenesis. Such variants include, for example, deletions from, and/orinsertions into and/or substitutions of, residues within the amino acidsequence of the polypeptide of interest, referred to herein as “aminoacid residue alterations.” Thus, a variant HVR refers to a HVRcomprising a variant sequence with respect to a starting or referencepolypeptide sequence (such as that of a source antibody or antigenbinding fragment). An amino acid residue alteration, in this context,refers to an amino acid different from the amino acid at thecorresponding position in a starting or reference polypeptide sequence(such as that of a reference antibody or fragment thereof). Anycombination of deletion, insertion, and substitution may be made toarrive at the final variant or mutant construct, provided that the finalconstruct possesses the desired functional characteristics. The aminoacid changes also may alter post-translational processes of thepolypeptide, such as changing the number or position of glycosylationsites.

A “wild-type (WT)” or “reference” sequence or the sequence of a“wild-type” or “reference” protein/polypeptide, such as an HVR or avariable domain of a reference antibody, may be the reference sequencefrom which variant polypeptides are derived through the introduction ofmutations. In general, the “wild-type” sequence for a given protein isthe sequence that is most common in nature. Similarly, a “wild-type”gene sequence is the sequence for that gene which is most commonly foundin nature. Mutations may be introduced into a “wild-type” gene (and thusthe protein it encodes) either through natural processes or throughman-induced means. The products of such processes are “variant” or“mutant” forms of the original “wild-type” protein or gene.

By “isoelectric point (pI)” is meant the pH at which a molecule (e.g., aprotein, such as an antibody) carries no net electrical charge, alsoreferred to in the art as “pH(I)” or “IEP.”

As used herein, an “antibody conjugate” is an antibody covalentlyattached to one or more polymers. Any suitable polymer may be conjugatedto an antibody, for example, a hydrophilic polymer (e.g., hyaluronicacid (HA) or polyethylene glycol (PEG)) or a hydrophobic polymer (e.g.,poly(lactic-co-glycolic acid) (PLGA)). In particular embodiments, thepolymer is HA (also referred to herein as “HA conjugates”).

As used herein, the term “polymer” means a molecule that includesrepeating structural units (i.e., monomers) connected by chemical bondsin a linear, circular, branched, crosslinked, or dendrimeric manner, ora combination thereof. A polymer may be synthetic or naturallyoccurring, or a combination thereof. It is to be understood that theterm “polymer” encompasses copolymers, which are polymers that includetwo or more different monomers. A polymer may also be a homopolymer,which is a polymer that includes only a single type of monomer.

The term “polydispersity index (PDI)” refers to a measure of thebroadness of the molecular weight distribution of a polymer. PDI is alsoreferred to in the art as “dispersity index,” “heterogeneity index,” or“dispersity (Ð).” The PDI of a polymer sample may be calculated usingequation (I): Ð_(M)=M_(w)/M_(n) where M_(w) is the weight-average molarmass and M_(n) is the number-average molar mass. Unless indicatedotherwise, PDI is calculated according to equation (I).

A polymer sample may be considered “monodisperse” (also known in the artas uniform) or “polydisperse” (also known in the art as non-uniform). Asused herein, the term “monodisperse” with respect to an HA polymer or HAconjugate sample means that the HA polymer or HA conjugate sample has aPDI of less than or equal to about 1.1, e.g., about 1.001, about 1.02,about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08,about 1.09, or about 1.1. For example, a monodisperse HA polymer or HAconjugate sample may have a PDI between 1.0 to about 1.1 (e.g., between1 to about 1.1, between 1 to about 1.09, between 1 to about 1.08,between 1 to about 1.07, between 1 to about 1.06, between 1 to about1.05, between 1 to about 1.04, between 1 to about 1.03, between 1 toabout 1.02, between 1 to about 1.01, between 1 to about 1.005, betweenabout 1.001 to about 1.1, between about 1.001 to about 1.1, betweenabout 1.001 to about 1.09, between about 1.001 to about 1.08, betweenabout 1.001 to about 1.07, between about 1.001 to about 1.06, betweenabout 1.001 to about 1.05, between about 1.001 to about 1.04, betweenabout 1.001 to about 1.03, between about 1.001 to about 1.02, betweenabout 1.001 to about 1.01, between about 1.001 to about 1.005, betweenabout 1.001 to about 1.004, between about 1.001 to about 1.003, betweenabout 1.001 to about 1.002, between about 1.0001 to about 1.1, betweenabout 1.0001 to about 1.09, between about 1.0001 to about 1.08, betweenabout 1.0001 to about 1.07, between about 1.0001 to about 1.06, betweenabout 1.0001 to about 1.05, between about 1.0001 to about 1.04, betweenabout 1.0001 to about 1.03, between about 1.0001 to about 1.02, betweenabout 1.0001 to about 1.01, between about 1.0001 to about 1.005, betweenabout 1.0001 to about 1.004, between about 1.0001 to about 1.003,between about 1.0001 to about 1.002, or between about 1.0001 to about1.005).

In contrast, the term “polydisperse” means that the HA polymer or HAconjugate sample has a PDI of greater than 1.1, e.g., about 1.3, about1.4, about 1.5, about 1.6, about 1.7, about 1.8, or higher. For example,in some embodiments, a polydisperse HA polymer or HA conjugate samplehas a PDI of between about 1.3 to about 2, about 1.4 to about 2, about1.5 to about 2, about 1.6 to about 2, about 1.7 to about 2, about 1.8 toabout 2, or about 1.9 to about 2.

The terms “hyaluronic acid,” “hyaluranon,” and “HA,” which are usedinterchangeably herein, refer to a polymeric glycosaminoglycan (GAG),which contains repeating disaccharide units of N-acetyl glucosamine andglucuronic acid. HA is an anionic, nonsulfated GAG, which can be found,for example, in extracellular matrix (e.g., in the vitreous of the eye),connective tissue, epithelial, and neural tissue.

The term “polyethylene glycol” or “PEG” as used herein, refers to apolyether compound that is also known as polyethylene oxide (PEO) orpolyoxyethylene (POE), depending on its molecular weight. PEG may have astructure of H—(O—CH₂—CH₂)_(n)—OH, wherein n is any suitable integer.The PEG may be a branched PEG, a star PEG, or a comb PEG. The PEG maybe, for example, a PEG tetramer, a PEG hexamer, or a PEG octamer.

The term “clearance,” as used herein, refers to the volume of asubstance (e.g., an anti-VEGF antibody, an antibody conjugate, a fusionprotein (e.g., a Fab fusion protein), or a polymeric formulation)cleared from a compartment (e.g., the eye (e.g., the vitreous)) per unittime.

The term “half-life” refers to the time required for the concentrationof a substance (e.g., an anti-VEGF antibody, an antibody conjugate, afusion protein (e.g., a Fab fusion protein), or a polymeric formulation)to decrease by one-half, in vivo (e.g., in the eye (e.g., the vitreous))or in vitro.

II. Compositions and Methods

The invention provides antibody conjugates that include polymers (e.g.,monodisperse polymers, including monodisperse HA polymers) linked toantibodies (e.g., anti-VEGF antibodies, including any anti-VEGF antibodydescribed herein), cysteine engineered antibodies that can be used, forexample, in preparing antibody conjugates, compositions that includeantibody conjugates (e.g., pharmaceutical compositions), as well asmethods of making and using the same, for example, for therapeutic uses(e.g., treatment of ocular disorders).

A. Exemplary Antibodies for Use in Conjugates of the Invention

The invention provides antibody conjugates that include antibodies(e.g., anti-VEGF antibodies) covalently linked to polymers (e.g.,monodisperse polymers). Any suitable antibody (e.g., anti-VEGF antibody)may be used. For example, the antibody may specifically bind to anantigen selected from the group consisting of VEGF; interleukin-1 beta(IL-1β); interleukin-6 (IL-6); interleukin-6 receptor (IL-6R);interleukin-13 (IL-13); IL-13 receptor (IL-13R); PDGF (e.g., PDGF-BB);angiopoietin; angiopoietin 2 (Ang2); Tie2; S1P; integrins αvβ3, αvβ5,and α5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D;TNFα; HtrA1; a VEGF receptor (e.g., VEGFR1, VEGFR2, VEGFR3,membrane-bound VEGF-receptor (mbVEGFR), or soluble VEGF receptor(sVEGFR)); ST-2 receptor; and a protein genetically linked toage-related macular degeneration (AMD) risk (e.g., complement pathwaycomponents C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1;ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC;COL10A1; and TNFRSF10A). Such antibodies can be useful, for example, forreducing angiogenesis and/or for treating or delaying the progression ofa disorder associated with pathological angiogenesis (e.g., oculardisorders). Exemplary, non-limiting anti-VEGF antibodies that can beused in the antibody conjugates of the invention are described furtherbelow.

In some instances, the anti-VEGF antibody may include at least one, two,three, four, five, or six HVRs selected from: (a) an HVR-H1 comprisingthe amino acid sequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2comprising the amino acid sequence of GX₁TPX₂GGX₃X₄X₅YX₆DSVX₇X₈ (SEQ IDNO: 2), wherein X₁ is Ile or His, X₂ is Ala or Arg, X₃ is Tyr or Lys, X₄is Thr or Glu, X₅ is Arg, Tyr, Gln, or Glu, X₆ is Ala or Glu, X₇ is Lysor Glu, and X₈ is Gly or Glu; (c) an HVR-H3 comprising the amino acidsequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising theamino acid sequence of RASQX₁VSTAVA (SEQ ID NO: 4), wherein X₁ is Asp orArg; (e) an HVR-L2 comprising the amino acid sequence of X₁ASFLYS (SEQID NO: 5), wherein X₁ is Ser or Met; and (f) an HVR-L3 comprising theamino acid sequence of X₁QGYGX₂PFT (SEQ ID NO: 6), wherein X₁ is Gln,Asn, or Thr and X₂ is Ala, Asn, Gln, or Arg, or a combination of one ormore of the above HVRs and one or more variants thereof having at leastabout 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity)to any one of SEQ ID NOs: 1-6.

For instance, the anti-VEGF antibody may include at least one, two,three, four, five, or six HVRs selected from: (a) an HVR-H1 comprisingthe amino acid sequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2comprising the amino acid sequence of GITPAGGYTRYADSVKG (SEQ ID NO: 7),GITPAGGYEYYADSVKG (SEQ ID NO: 21), or GITPAGGYEYYADSVEG (SEQ ID NO: 22);(c) an HVR-H3 comprising the amino acid sequence of FVFFLPYAMDY (SEQ IDNO: 3); (d) an HVR-L1 comprising the amino acid sequence of RASQDVSTAVA(SEQ ID NO: 8); (e) an HVR-L2 comprising the amino acid sequence ofSASFLYS (SEQ ID NO: 9); and (f) an HVR-L3 comprising the amino acidsequence of QQGYGAPFT (SEQ ID NO: 10) or QQGYGNPFT (SEQ ID NO: 23), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1, 3, 7-10, or21-23.

For example, in some instances, the anti-VEGF antibody may include atleast one, two, three, four, five, or six HVRs selected from: (a) anHVR-H1 comprising the amino acid sequence of DYWIH (SEQ ID NO: 1); (b)an HVR-H2 comprising the amino acid sequence of GITPAGGYTRYADSVKG (SEQID NO: 7); (c) an HVR-H3 comprising the amino acid sequence ofFVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2 comprising theamino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1, 3, or 7-10. In aparticular example, in some instances, the anti-VEGF antibody includesthe following six HVRs: (a) an HVR-H1 comprising the amino acid sequenceof DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acidsequence of GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) an HVR-H3 comprisingthe amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) anHVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and(f) an HVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ IDNO: 10).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): (a) an FR-H1 comprising the amino acid sequenceof EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC(SEQ ID NO: 19); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20).

For example, in some instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 11 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 12.

For example, in some instances, the anti-VEGF antibody may include atleast one, two, three, four, five, or six HVRs selected from: (a) anHVR-H1 comprising the amino acid sequence of DYWIH (SEQ ID NO: 1); (b)an HVR-H2 comprising the amino acid sequence of GITPAGGYEYYADSVEG (SEQID NO: 22); (c) an HVR-H3 comprising the amino acid sequence ofFVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2 comprising theamino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3comprising the amino acid sequence of QQGYGNPFT (SEQ ID NO: 23), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1, 3, 8, 9, 22, or23. In a particular example, in some instances, the anti-VEGF antibodyincludes the following six HVRs: (a) an HVR-H1 comprising the amino acidsequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the aminoacid sequence of GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3comprising the amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) anHVR-L1 comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8);(e) an HVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO:9); and (f) an HVR-L3 comprising the amino acid sequence of QQGYGNPFT(SEQ ID NO: 23).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): (a) an FR-H1 comprising the amino acid sequenceof EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) an FR-H3comprising the amino acid sequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR(SEQ ID NO: 31); and (d) an FR-H4 comprising the amino acid sequence ofWGQGELVTVSS (SEQ ID NO: 32).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 24); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20).

For example, in some instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGNPFT (SEQ ID NO: 23).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 38.

In some instances, the anti-VEGF antibody includes the following sixHVRs: (a) an HVR-H1 comprising the amino acid sequence of DYWIH (SEQ IDNO: 1); (b) an HVR-H2 comprising the amino acid sequence ofGITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGNPFT (SEQ ID NO: 23).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 38.

For example, in some instances, the anti-VEGF antibody may include atleast one, two, three, four, five, or six HVRs selected from: (a) anHVR-H1 comprising the amino acid sequence of DYWIH (SEQ ID NO: 1); (b)an HVR-H2 comprising the amino acid sequence of GITPAGGYEYYADSVEG (SEQID NO: 22); (c) an HVR-H3 comprising the amino acid sequence ofFVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acidsequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2 comprising theamino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) an HVR-L3comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10), or acombination of one or more of the above HVRs and one or more variantsthereof having at least about 80% sequence identity (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to any one of SEQ ID NOs: 1, 3, 8-10, or 22.In a particular example, in some instances, the anti-VEGF antibodyincludes the following six HVRs: (a) an HVR-H1 comprising the amino acidsequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the aminoacid sequence of GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3comprising the amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) anHVR-L1 comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8);(e) an HVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO:9); and (f) an HVR-L3 comprising the amino acid sequence of QQGYGAPFT(SEQ ID NO: 10).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): (a) an FR-H1 comprising the amino acid sequenceof EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30); (c) an FR-H3comprising the amino acid sequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR(SEQ ID NO: 31); and (d) an FR-H4 comprising the amino acid sequence ofWGQGELVTVSS (SEQ ID NO: 32).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ IDNO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18) orWYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19) orGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20).

For example, in some instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 34.

For example, in other instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGEAPKLLIY (SEQ ID NO: 27);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 35.

For example, in other instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGEAPKLLIY (SEQ ID NO: 27);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 35.

For example, in yet other instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPESLSASVGDEVTITC (SEQ ID NO: 25); (b) an FR-L2comprising the amino acid sequence of WYQQKPGEAPKLLIY (SEQ ID NO: 27);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 36.

For example, in still further instances, the anti-VEGF antibody includesthe following six HVRs: (a) an HVR-H1 comprising the amino acid sequenceof DYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acidsequence of GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprisingthe amino acid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1comprising the amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) anHVR-L2 comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and(f) an HVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ IDNO: 10). In some instances, the anti-VEGF antibody includes thefollowing four heavy chain variable domain FRs: (a) an FR-H1 comprisingthe amino acid sequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO:29); (b) an FR-H2 comprising the amino acid sequence of WVRQEPGEGLEWVA(SEQ ID NO: 30); (c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 37.

In other instances, the anti-VEGF antibody includes the following sixHVRs: (a) an HVR-H1 comprising the amino acid sequence of DYWIH (SEQ IDNO: 1); (b) an HVR-H2 comprising the amino acid sequence ofGITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 37.

For example, in other instances, the anti-VEGF antibody includes thefollowing six HVRs: (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 12.

In other instances, the anti-VEGF antibody includes the following sixHVRs: (a) an HVR-H1 comprising the amino acid sequence of DYWIH (SEQ IDNO: 1); (b) an HVR-H2 comprising the amino acid sequence ofGITPAGGYEYYADSVEG (SEQ ID NO: 22); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following fourheavy chain variable domain FRs: (a) an FR-H1 comprising the amino acidsequence of EEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2comprising the amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) an FR-H4comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32). Infurther instances, the anti-VEGF antibody includes the following fourlight chain variable domain FRs: (a) an FR-L1 comprising the amino acidsequence of DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18);(c) an FR-L3 comprising the amino acid sequence ofGVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20). Insome instances, the anti-VEGF antibody includes (a) a VH domaincomprising an amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising an amino acid sequence of SEQ ID NO: 12.

In some instances, the anti-VEGF antibody comprises (a) a heavy chainvariable (VH) domain comprising an amino acid sequence having at least90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity) to, or the sequence of, any one of SEQ IDNOs: 11, 40, or 42; (b) a light chain variable (VL) domain comprising anamino acid sequence having at least 90% sequence (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to, or thesequence of, any one of SEQ ID NOs: 12, 41, or 46; or (c) a VH domain asin (a) and a VL domain as in (b). For example, in some instances, theantibody comprises a VH domain comprising the amino acid sequence of SEQID NO: 11 and a VL domain comprising the amino acid sequence of SEQ IDNO: 12. In some instances, the antibody comprises a VH domain comprisingthe amino acid sequence of SEQ ID NO: 40 and a VL domain comprising theamino acid sequence of SEQ ID NO: 12. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:42 and a VL domain comprising the amino acid sequence of SEQ ID NO: 12.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 42 and a VL domain comprising theamino acid sequence of SEQ ID NO: 41. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:11 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46.

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): (a) an FR-H1 comprising the amino acid sequenceof EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14) orWVRQEPGKGLEWVA (SEQ ID NO: 39); (c) an FR-H3 comprising the amino acidsequence of RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) anFR-H4 comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17) or DIQMTQSPSSLSASVGDRVTIDC (SEQID NO: 45); (b) an FR-L2 comprising the amino acid sequence ofWYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19),GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID NO: 44), orGVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 54); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20) orFGQGTKVEVK (SEQ ID NO: 55).

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 11 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 59) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQ GTKVEIK.

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 59) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQ GTKVEIK.

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 40 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 59) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQ GTKVEIK.

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 42 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 59) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQ GTKVEIK.

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 51 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 59) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGNPFTFGQ GTKVEIK.

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 11 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQ GTKVEIK.

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQ GTKVEIK.

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 40 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQ GTKVEIK.

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 42 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQ GTKVEIK.

In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 51 and a VL domain comprising theamino acid sequence of

(SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQGYGAPFTFGQ GTKVEIK.For example, in some instances, the anti-VEGF antibody comprises (a) aVH domain comprising an amino acid sequence having at least 90% sequenceidentity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity) to, or the sequence of, SEQ ID NO: 11; (b) a VLdomain comprising an amino acid sequence having at least 90% sequence(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO: 11; or (c) a VH domain asin (a) and a VL domain as in (b). In some instances, the anti-VEGFantibody may include (a) an HVR-H1 comprising the amino acid sequence ofDYWIH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequenceof GITPAGGYTRYADSVKG (SEQ ID NO: 7); (c) an HVR-H3 comprising the aminoacid sequence of FVFFLPYAMDY (SEQ ID NO: 3); (d) an HVR-L1 comprisingthe amino acid sequence of RASQDVSTAVA (SEQ ID NO: 8); (e) an HVR-L2comprising the amino acid sequence of SASFLYS (SEQ ID NO: 9); and (f) anHVR-L3 comprising the amino acid sequence of QQGYGAPFT (SEQ ID NO: 10).In some instances, the anti-VEGF antibody includes the following heavychain framework regions: (a) an FR-H1 comprising the amino acid sequenceof EVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2comprising the amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14);(c) an FR-H3 comprising the amino acid sequence ofRFTISADTSKNTAYLQMRSLRAEDTAVYYCAR (SEQ ID NO: 15); and (d) an FR-H4comprising the amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 16). Insome instances, the anti-VEGF antibody includes the following lightchain framework regions: (a) an FR-L1 comprising the amino acid sequenceof DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC(SEQ ID NO: 19); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20). In some instances, the anti-VEGF antibodyincludes a binding domain comprising (a) a VH domain comprising an aminoacid sequence of SEQ ID NO: 11 and (b) a VL domain comprising an aminoacid sequence of SEQ ID NO: 12. In some instances, the exemplaryanti-VEGF is N94A.F83A.N82aR.Y58R (also referred to as G6.31 AARR orG6.31.AARR).

In some instances, the anti-VEGF antibody comprises (a) VH domaincomprising an amino acid sequence having at least 90% sequence identity(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO: 33 or 51; (b) a VL domaincomprising an amino acid sequence having at least 90% sequence (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity)to, or the sequence of, any one of SEQ ID NOs: 12, 34, 35, 36, 37, or38; or (c) a VH domain as in (a) and a VL domain as in (b). For example,in some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence of SEQ ID NO: 12. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 34.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence of SEQ ID NO: 35. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 36.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 33 and a VL domain comprising theamino acid sequence of SEQ ID NO: 37. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:33 and a VL domain comprising the amino acid sequence of SEQ ID NO: 38.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 51 and a VL domain comprising theamino acid sequence of SEQ ID NO: 38. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:51 and a VL domain comprising the amino acid sequence of SEQ ID NO: 35.In some instances, the antibody comprises a VH domain comprising theamino acid sequence of SEQ ID NO: 51 and a VL domain comprising theamino acid sequence of SEQ ID NO: 37. In some instances, the antibodycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:51 and a VL domain comprising the amino acid sequence of SEQ ID NO: 12.

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following heavy chain variable domainframework regions (FRs): an FR-H1 comprising the amino acid sequence ofEEQLVEEGGGLVQPGESLELSCAASGFEIS (SEQ ID NO: 29) orEEQLVEEGGGLVQPGESLRLSCAASGFEIS (SEQ ID NO: 52); (b) an FR-H2 comprisingthe amino acid sequence of WVRQEPGEGLEWVA (SEQ ID NO: 30) orWVRQEPGKGLEWVA (SEQ ID NO: 39); (c) an FR-H3 comprising the amino acidsequence of RFTISADTSENTAYLQMNELRAEDTAVYYCAR (SEQ ID NO: 31); and (d) anFR-H4 comprising the amino acid sequence of WGQGELVTVSS (SEQ ID NO: 32).

In some instances, any of the preceding anti-VEGF antibodies may includeone, two, three, or four of the following light chain variable domainFRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17), DIQMTQSPESLSASVGDEVTITC (SEQ IDNO: 25), or DIQMTQSPSSLSASVGDEVTITC (SEQ ID NO: 26); (b) an FR-L2comprising the amino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18) orWYQQKPGEAPKLLIY (SEQ ID NO: 27); (c) an FR-L3 comprising the amino acidsequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 19),GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24), orGVPSRFSGSGSGTDFTLTIESLQPEDAATYYC (SEQ ID NO: 28); and (d) an FR-L4comprising the amino acid sequence of FGQGTKVEIK (SEQ ID NO: 20).

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 48 and/or(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.In certain embodiments, the antibody is G6.31 AARR expressed in Fabformat.

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 49 and/or(b) a light chain comprising the amino acid sequence of SEQ ID NO: 50.In certain embodiments, the antibody is a variant version of G6.31 AARRthat lacks reactivity to anti-human IgG.

In a further aspect, an antibody (e.g., an anti-VEGF antibody) accordingto any of the above embodiments may incorporate any of the features,singly or in combination, as described in Sections 1-8 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g., 10⁻⁹ M or less, e.g., from 10⁻⁹ M to 10⁻¹³ M, e.g.,from 10⁻⁹M to 10⁻¹³ M). For example, in some instances, an antibodyprovided herein binds an antigen (e.g., human VEGF (hVEGF)) with a Kd ofabout 10 nM or lower. In some instances, an antibody provided hereinbinds an antigen (e.g., hVEGF) with a Kd of about 5 nM or lower. In someinstances, an antibody provided herein binds hVEGF with a Kd of about 2nM or lower. For example, in some instances, the antibody binds anantigen (e.g., hVEGF) with a Kd between about 25 pM and about 2 nM(e.g., about 25 pM, about 50 pM, about 75 pM, about 100 pM, about 125pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM, about 250pM, about 275 pM, about 300 pM, about 325 pM, about 350 pM, about 375pM, about 400 pM, about 425 pM, about 450 pM, about 475 pM, about 500pM, about 525 pM, about 550 pM, about 575 pM, about 600 pM, about 625pM, about 650 pM, about 675 pM, about 700 pM, about 725 pM, about 750pM, about 775 pM, about 800 pM, about 825 pM, about 850 pM, about 875pM, about 900 pM, about 925 pM, about 950 pM, about 975 pM, about 1 nM,about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM,about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, or about 2 nM).In some instances, the antibody binds an antigen (e.g., hVEGF) with a Kdbetween about 75 pM and about 600 pM (e.g., about 75 pM, about 100 pM,about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM,about 250 pM, about 275 pM, about 300 pM, about 325 pM, about 350 pM,about 375 pM, about 400 pM, about 425 pM, about 450 pM, about 475 pM,about 500 pM, about 525 pM, about 550 pM, about 575 pM, about 600 pM).In some instances, the antibody binds an antigen (e.g., hVEGF) with a Kdbetween about 75 pM and about 500 pM. In some instances, the antibodybinds an antigen (e.g., hVEGF) with a Kd between about 75 pM and about400 pM. In some instances, the antibody binds an antigen (e.g., hVEGF)with a Kd between about 75 pM and about 300 pM. In some instances, theantibody binds an antigen (e.g., hVEGF) with a Kd between about 75 pMand about 200 pM. In some instances, the antibody binds an antigen(e.g., hVEGF) with a Kd between about 75 pM and about 150 pM. In someinstances, the antibody binds an antigen (e.g., hVEGF) with a Kd betweenabout 75 pM and about 125 pM. In some instances, the antibody binds anantigen (e.g., hVEGF) with a Kd between about 75 pM and about 100 pM. Insome instances, the antibody binds an antigen (e.g., hVEGF) with a Kd ofabout 80 pM. In some instances, the antibody binds an antigen (e.g.,hVEGF) with a Kd of about 60 pM. In some instances, the antibody bindsan antigen (e.g., hVEGF) with a Kd of about 40 pM.

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA). In one embodiment, an RIA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881(1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin (BSA) in phosphate buffered saline (PBS) for two to fivehours at room temperature (approximately 23° C.). In a non-adsorbentplate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed withserial dilutions of a Fab of interest (e.g., consistent with assessmentof the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res.57:4593-4599 (1997)). The Fab of interest is then incubated overnight;however, the incubation may continue for a longer period (e.g., about 65hours) to ensure that equilibrium is reached. Thereafter, the mixturesare transferred to the capture plate for incubation at room temperature(e.g., for one hour). The solution is then removed and the plate washedeight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plateshave dried, 150 μl/well of scintillant (MICROSCINT-20™; Packard) isadded, and the plates are counted on a TOPCOUNT™ gamma counter (Packard)for ten minutes. Concentrations of each Fab that give less than or equalto 20% of maximal binding are chosen for use in competitive bindingassays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CM5 chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CM5, BIAcore,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, for example, Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Stability

In some instances, the antibody used in the antibody conjugates of theinvention or compositions thereof have enhanced stability, for example,as compared to an anti-VEGF antibody, for instance, G6.31 (see, e.g.,U.S. Pat. No. 7,758,859 and International Application Pub. No. WO2005/012359, which are incorporated herein by reference in theirentirety). The stability of an antibody may be determined using anymethod known in the art, for example, differential scanning fluorimetry(DSF), circular dichroism (CD), intrinsic protein fluorescence,differential scanning calorimetry, spectroscopy, light scattering (e.g.,dynamic light scattering (DLS) and static light scattering (SLS),self-interaction chromatography (SIC). The anti-VEGF antibody may have,for example, an enhanced melting temperature (Tm), temperature ofaggregation (T_(agg)), or other metrics of stability compared to ananti-VEGF antibody, for example, G6.31.

In certain embodiments, an antibody provided herein has a Tm that isgreater than or equal to about 80° C. (e.g., about 81° C., about 82° C.,about 83° C., about 84° C., about 85° C., about 86° C., about 87° C.,about 88° C., about 89° C., about 90° C., about 91° C., about 92° C., orabout 93° C.). For example, in some instances, the anti-VEGF antibodyhas a Tm that is greater than or equal to about 83.5° C. (e.g., about83.5° C., about 84° C., about 85° C., about 86° C., about 87° C., about88° C., about 89° C., about 90° C., about 91° C., about 92° C., or about93° C.). In some instances, the anti-VEGF antibody has a Tm of about 82°C. to about 92° C. (e.g., about 82° C., about 83° C., about 84° C.,about 85° C., about 86° C., about 87° C., about 88° C., about 89° C.,about 90° C., about 91° C., or about 92° C.). In some about instances,the anti-VEGF antibody has a Tm of about 82° C. In some instances, anyof the preceding Tm values of an anti-VEGF antibody is determined usingDSF. In some embodiments, the Tm value of an anti-VEGF antibody isdetermined as described, for example, in Example 1 of InternationalPatent Application No. PCT/US2016/053454, which is incorporated hereinby reference in its entirety.

3. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab-C, Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragmentsdescribed below. For a review of certain antibody fragments, see Hudsonet al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see,e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

4. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, for example, inU.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci.USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprisesa non-human variable region (e.g., a variable domain derived from amouse, rat, hamster, rabbit, or non-human primate, such as a monkey) anda human constant domain. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, for example, CDRs, (or portionsthereof) are derived from a non-human antibody, and FRs (or portionsthereof) are derived from human antibody sequences. A humanized antibodyoptionally will also comprise at least a portion of a human constantregion. In some embodiments, some FR residues in a humanized antibodyare substituted with corresponding residues from a non-human antibody(e.g., the antibody from which the HVR residues are derived), e.g., torestore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, forexample, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008),and are further described, for example, in Riechmann et al., Nature332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321,and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describingspecificity determining region (SDR) grafting); Padlan, Mol. Immunol.28:489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000)(describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal., J. Immunol, 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

5. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584 describingXENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB®technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology,and U.S. Patent Application Publication No. US 2007/0061900, describingVELOCIMOUSE® technology). Human variable regions from intact antibodiesgenerated by such animals may be further modified, for example, bycombining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

6. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments can be derived from phage libraries asdescribed in International Patent Application No. PCT/US2016/053454.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

7. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, for example, a bispecific antibody. Multispecific antibodiesare monoclonal antibodies that have binding specificities for at leasttwo different sites. In certain embodiments, one of the bindingspecificities is for VEGF and the other is for any other antigen (e.g.,a second biological molecule, e.g., interleukin-1 beta (IL-1β);interleukin-6 (IL-6); interleukin-6 receptor (IL-6R); interleukin-13(IL-13); IL-13 receptor (IL-13R); PDGF (e.g., PDGF-BB); angiopoietin;angiopoietin 2 (Ang2); Tie2; S1P; integrins αvβ3, αvβ5, and α5β1;betacellulin; apelin/APJ; erythropoietin; complement factor D; TNFα;HtrA1; a VEGF receptor (e.g., VEGFR1, VEGFR2, VEGFR3, membrane-boundVEGF-receptor (mbVEGFR), or soluble VEGF receptor (sVEGFR)); ST-2receptor; and proteins genetically linked to age-related maculardegeneration (AMD) risk, such as complement pathway components C2,factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3;HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; andTNFRSF10A. Accordingly, the bispecific antibody may have bindingspecificity for VEGF and IL-1β; VEGF and IL-6; VEGF and IL-6R; VEGF andIL-13; VEGF and IL-13R; VEGF and PDGF (e.g., PDGF-BB); VEGF andangiopoietin; VEGF and Ang2; VEGF and Tie2; VEGF and S1P; VEGF andintegrin αvβ3; VEGF and integrin αvβ5; VEGF and integrin α5β1; VEGF andbetacellulin; VEGF and apelin/APJ; VEGF and erythropoietin; VEGF andcomplement factor D; VEGF and TNFα; VEGF and HtrA1; VEGF and a VEGFreceptor (e.g., VEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR); VEGF andST-2 receptor; VEGF and C2; VEGF and factor B; VEGF and factor H; VEGFand CFHR3; VEGF and C3b; VEGF and C5; VEGF and C5a; VEGF and C3a; VEGFand ARMS2; VEGF and TIMP3; VEGF and HLA; VEGF and IL-8; VEGF and CX3CR1;VEGF and TLR3; VEGF and TLR4; VEGF and CETP; VEGF and LIPC; VEGF andCOL10A1; or VEGF and TNFRSF10A. In certain embodiments, bispecificantibodies may bind to two different epitopes of VEGF. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress VEGF. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments (e.g., Fab, Fab′, or Fab-C fragments).

In some instances, the bispecific antibody is a bispecificanti-VEGF/anti-angiopoeitin 2 (Ang2) antibody disclosed in U.S. PatentApplication No. US 2014/0017244, which is incorporated herein byreference in its entirety. For example, the anti-VEGF/anti-Ang2bispecific antibody may include a first binding domain that binds VEGF(such as any of the anti-VEGF antibodies described herein) and a secondbinding domain that binds Ang2 that includes (a) an HVR-H1 comprisingthe amino acid sequence of GYYMH (SEQ ID NO: 62); (b) an HVR-H2comprising the amino acid sequence of WINPNSGGTNYAQKFQG (SEQ ID NO: 63);(c) an HVR-H3 comprising the amino acid sequence of SPNPYYYDSSGYYYPGAFDI(SEQ ID NO: 64); (d) an HVR-L1 comprising the amino acid sequence ofGGNNIGSKSVH (SEQ ID NO: 65); (e) an HVR-L2 comprising the amino acidsequence of DDSDRPS (SEQ ID NO: 66); and (f) an HVR-L3 comprising theamino acid sequence of QVWDSSSDHWV (SEQ ID NO: 67), or a combination ofone or more of the above HVRs and one or more variants thereof having atleast about 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity) to any one of SEQ ID NOs: 62-67.

In some instances, the anti-VEGF/anti-Ang2 bispecific antibody mayinclude a first binding domain that binds VEGF (such as any of theanti-VEGF antibodies described herein) and a second binding domain thatbinds to Ang2 and includes (a) a VH domain comprising an amino acidsequence having at least 80% sequence identity (e.g., 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity) to, or the sequence of, SEQ ID NO:68; (b) a VL domain comprising an amino acid sequence having at least80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity) to, or the sequence of, SEQ ID NO: 69; or (c) a VH domain asin (a) and a VL domain as in (b). In some instances, theanti-VEGF/anti-Ang2 bispecific antibody may include a first bindingdomain that binds VEGF (such as any of the anti-VEGF antibodiesdescribed herein) and a second binding domain that specifically bind toAng2, wherein the second binding domain is any antibody binding domaindescribed in International Patent Application Publication No. WO2010/069532, which is incorporated herein by reference in its entirety,or a variant thereof.

In other instances, the anti-VEGF/anti-Ang2 bispecific antibody is anyanti-VEGF/anti-Ang2 bispecific antibody described in InternationalPatent Application Publication No. WO 2016/073157.

In some instances, the bispecific antibody is a bispecificanti-VEGF/anti-IL-6 antibody. In some instances, an anti-VEGF/anti-IL-6bispecific antibody may include a first binding domain that binds VEGF(such as any of the anti-VEGF antibodies described herein) and a secondbinding domain that binds IL-6. The second binding domain may be abinding domain of any anti-IL-6 antibody known in the art, for example,EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890, which isincorporated herein by reference in its entirety), siltuximab(SYLVANT®), olokizumab, clazakizumab, sirukumab, elsilimomab,gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof.

In some instances, the bispecific antibody is a bispecificanti-VEGF/anti-IL-6R antibody. In some instances, ananti-VEGF/anti-IL-6R bispecific antibody may include a first bindingdomain that binds VEGF (such as any of the anti-VEGF antibodiesdescribed herein) and a second binding domain that binds IL-6R. Thesecond binding domain may be a binding domain any anti-IL-6R antibodyknown in the art, for example, tocilizumab (ACTEMRA®) (see, e.g., WO1992/019579, which is incorporated herein by reference in its entirety),sarilumab, vobarilizumab (ALX-0061), SA-237, or a variant thereof.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, for example, in Tutt etal., J. Immunol. 147:60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to VEGF as well asanother, different antigen (see, e.g., US 2008/0069820).

8. Antibody Variants

In certain embodiments, amino acid sequence variants (e.g., antibodyvariants including one or more amino acid residue alterations) of theantibodies provided herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of an antibodymay be prepared by introducing appropriate modifications into thenucleotide sequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of residues within the amino acid sequences ofthe antibody. Any combination of deletion, insertion, and substitutioncan be made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, for example, antigenbinding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, for example, retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Original Residue Exemplary Substitutions Preferred SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues and/or FR residues of a parent antibody(e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further study will have modifications (e.g.,improvements) in certain biological properties (e.g., increasedaffinity, increased stability, increased expression, altered pI, and/orreduced immunogenicity) relative to the parent antibody and/or will havesubstantially retained certain biological properties of the parentantibody. An exemplary substitutional variant is an affinity maturedantibody, which may be conveniently generated, for example, using phagedisplay-based affinity maturation techniques such as those describedherein. Briefly, one or more HVR residues are mutated and the variantantibodies displayed on phage and screened for a particular biologicalactivity (e.g. binding affinity).

Alterations (e.g., substitutions) may be made in HVRs, for example, toimprove antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation athigh frequency during the somatic maturation process (see, e.g.,Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues thatcontact antigen, with the resulting variant VH or VL being tested forbinding affinity. Affinity maturation by constructing and reselectingfrom secondary libraries has been described, for example, in Hoogenboomet al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed.,Human Press, Totowa, N.J., (2001)). In some embodiments of affinitymaturation, diversity is introduced into the variable genes chosen formaturation by any of a variety of methods (e.g., error-prone PCR, chainshuffling, or oligonucleotide-directed mutagenesis). A secondary libraryis then created. The library is then screened to identify any antibodyvariants with the desired affinity. Another method to introducediversity involves HVR-directed approaches, in which several HVRresidues (e.g., 4-6 residues at a time) are randomized. HVR residuesinvolved in antigen binding may be specifically identified, e.g., usingalanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 inparticular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more FRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Suchalterations may, for example, improve antibody affinity and/or stability(e.g., as assessed by an increased melting temperature).

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, for example, US Patent Publication Nos. US 2003/0157108; US2004/0093621. Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al., J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614(2004). Examples of cell lines capable of producing defucosylatedantibodies include Led 3 CHO cells deficient in protein fucosylation(Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Pat ApplNo US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No.6,602,684; and US 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants may have improved CDCfunction. Such antibody variants are described, for example, in WO1997/30087; WO 1998/58964; and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid residue alteration (e.g., asubstitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).

Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337; and Bruggemann et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCYTOTOX 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, for example, in an animal model such as that disclosedin Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1qbinding assays may also be carried out to confirm that the antibody isunable to bind C1q and hence lacks CDC activity. See, for example, C1qand C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assesscomplement activation, a CDC assay may be performed (see, e.g.,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg etal., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743(2004)). FcRn binding and in vivo clearance/half life determinations canalso be performed using methods known in the art (see, e.g., Petkova etal., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)). In certainembodiments, an antibody variant comprises an Fc region with one or moreamino acid substitutions which improve ADCC, e.g., substitutions atpositions 298, 333, and/or 334 of the Fc region (EU numbering ofresidues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), for example, as described inU.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol.164: 4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

The invention provides cysteine engineered antibodies where one or moreamino acids of a wild-type or parent antibody (e.g., an anti-VEGFantibody, including any anti-VEGF antibody described herein) arereplaced (i.e., “substituted” or “mutated”) with a cysteine amino acid(i.e., an “engineered cysteine”). Any form of antibody may be soengineered, i.e. mutated. For example, a parent monoclonal antibody maybe engineered to form a “THIOMAB™ antibody.” One example of a THIOMAB™antibody is an antibody fragment (i.e., a Fab) that has an engineeredcysteine. This Fab THIOMAB™ antibody can be referred to as “ThioFab.” Itshould be noted that a single site mutation yields a single engineeredcysteine residue in a ThioFab, while a single site mutation yields twoengineered cysteine residues in a THIOMAB™ antibody, due to the dimericnature of the IgG antibody.

Mutants with engineered cysteine (Cys) residues can be evaluated for thereactivity of the newly introduced, engineered cysteine thiol groups.The thiol reactivity value is a relative, numerical term in the range of0 to 1.0 and can be measured for any cysteine engineered antibody. Thiolreactivity values of cysteine engineered antibodies of the invention arein the range of 0.0 to 1.0. Specifically, the thiol reactivity values ofcysteine engineered antibodies of the invention are in the range of 0.1to 1.0. In certain embodiments, the thiol reactivity values of cysteineengineered antibodies of the invention are in the ranges of 0.0 to 0.1,0.1 to 0.5, 0.1 to 0.6, 0.1 to 0.7, 0.1 to 0.8, 0.1 to 0.9, or 0.1 to1.0. In certain embodiments, the thiol reactivity values of cysteineengineered antibodies of the invention are in the ranges of 0.2 to 1.0,0.3 to 1.0, 0.4 to 1.0, 0.5 to 1.0, 0.6 to 1.0, 0.7 to 1.0, or 0.8 to1.0. In certain embodiments, the thiol reactivity values of cysteineengineered antibodies of the invention are in the range of 0.6 to 1.0.In certain embodiments, the thiol reactivity values of cysteineengineered antibodies of the invention are in the ranges of 0.7 to 1.0.In certain embodiments, the thiol reactivity values of cysteineengineered antibodies of the invention are in the ranges of 0.8 to 10.In certain embodiments, the thiol reactivity values of cysteineengineered antibodies of the invention are in the ranges of 0.5 to 0.8.In certain embodiments, the thiol reactivity values of cysteineengineered antibodies of the invention are in the ranges of 0.5 to 0.9.In certain embodiments, the thiol reactivity values of cysteineengineered antibodies of the invention are in the ranges of 0.5 to 0.7.In certain embodiments, the thiol reactivity values of cysteineengineered antibodies of the invention are in the ranges of 0.5 to 1.0.

The invention provides design, selection, and preparation methods forproducing cysteine engineered antibodies which are reactive withelectrophilic functionality. These methods further enable antibodyconjugate compounds such as, for example, antibody-polymer conjugateswith polymer moieties conjugated at designated, designed, selectivesites. Reactive cysteine residues on an antibody surface allowspecifically conjugating a polymer through a thiol reactive group suchas maleimide or haloacetyl. The nucleophilic reactivity of the thiolfunctionality of a Cys residue to a maleimide group is about 1000 timeshigher compared to any other amino acid functionality in a protein, suchas amino group of lysine residues or the N-terminal amino group. Thiolspecific functionality in iodoacetyl and maleimide reagents may reactwith amine groups, but higher pH (>9.0) and longer reaction times arerequired (Garman, 1997, Non-Radioactive Labelling: A Practical Approach,Academic Press, London).

Cysteine engineered antibodies of the invention preferably retain theantigen binding capability of their wild type, parent antibodycounterparts. Thus, cysteine engineered antibodies are capable ofbinding, preferably specifically, to antigens. Exemplary, non-limitingantigens include VEGF; IL-1β; IL-6; IL-6R; IL-13; IL-13R; PDGF (e.g.,PDGF-BB); angiopoietin; angiopoietin 2 (Ang2); Tie2; S1P; integrinsαvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin;complement factor D; TNFα; HtrA1; a VEGF receptor (e.g., VEGFR1, VEGFR2,VEGFR3, mbVEGFR, or sVEGFR); ST-2 receptor; and a protein geneticallylinked to AMD risk (e.g., complement pathway components C2, factor B,factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8;CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A).

Any of the antibodies described herein (e.g., any of the anti-VEGFantibodies described above) may be a parent antibody used to generate acysteine engineered antibody. The exemplary methods described here maybe applied generally to the identification and production of antibodiesthrough application of the design and screening steps described herein.

Cysteine engineered antibodies of the invention may be site-specificallyand efficiently coupled with a thiol-reactive reagent. Thethiol-reactive reagent may be, for example, a clearance-modifying agentsuch as a polymer (e.g., an HA polymer or various isomers ofpolyethylene glycol), a peptide that binds to a third component, oranother carbohydrate or lipophilic agent, a multifunctional linkerreagent, a capture, i.e., affinity, label reagent (e.g., a biotin-linkerreagent), a detection label (e.g., a fluorophore reagent), a solid phaseimmobilization reagent (e.g., SEPHAROSE™, polystyrene, or glass), or adrug-linker intermediate. One example of a thiol-reactive reagent isN-ethyl maleimide (NEM). In an exemplary embodiment, reaction of aTHIOMAB™ antibody with a biotin-linker reagent provides a biotinylatedTHIOMAB™ antibody by which the presence and reactivity of the engineeredcysteine residue may be detected and measured. Reaction of a THIOMAB™antibody with a multifunctional linker reagent provides a THIOMAB™antibody with a functionalized linker which may be further reacted witha polymer, a drug moiety reagent, or other label. Reaction of a THIOMAB™antibody with a drug-linker intermediate provides a THIOMAB™ antibodydrug conjugate. In certain embodiments, the THIOMAB™ antibody is aThioFab.

Cysteine engineered antibodies can be conjugated to thiol-reactiveagents in which the reactive group is, for example, a maleimide, aniodoacetamide, a pyridyl disulfide, or other thiol-reactive conjugationpartner (see, e.g., Haugland, 2003, Molecular Probes Handbook ofFluorescent Probes and Research Chemicals, Molecular Probes, Inc.;Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-RadioactiveLabelling: A Practical Approach, Academic Press, London; Means (1990)Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996)Academic Press, San Diego, pp. 40-55, 643-671). The partner may be acytotoxic agent (e.g., a toxin such as doxorubicin or pertussis toxin),a fluorophore such as a fluorescent dye like fluorescein or rhodamine, achelating agent for an imaging or radiotherapeutic metal, a peptidyl ornon-peptidyl label or detection tag, or a clearance-modifying agent suchas a polymer (e.g., an HA polymer or various isomers of polyethyleneglycol), a peptide that binds to a third component, or anothercarbohydrate or lipophilic agent.

The PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) assayallows for detection of reactive cysteine groups in antibodies in anELISA phage format. See U.S. Pat. No. 7,521,541 and U.S. Pat. Pub. No.20110301334, which are incorporated herein by reference in theirentirety. Specifically, the PHESESLECTOR assay includes the process ofcoating the protein (e.g., antibody) of interest on well surfaces,followed incubation with phage particles and then horseradish peroxidase(HRP) labeled secondary antibody with absorbance detection. Mutantproteins displayed on phage may be screened in a rapid, robust, andhigh-throughput manner. Libraries of cysteine engineered antibodies canbe produced and subjected to binding selection using the same approachto identify appropriately reactive sites of free Cys incorporation fromrandom protein-phage libraries of antibodies or other proteins. Thistechnique includes reacting cysteine mutant proteins displayed on phagewith an affinity reagent or reporter group which is also thiol-reactive.

In certain embodiments, the PHESELECTOR assay includes the followingsteps: (1) bovine serum albumin (BSA), a portion or entirety of a targetprotein (e.g., VEGF), and streptavidin (100 μl of 2 μg/ml) areseparately coated on MAXISORB® 96 well plates; (2) After blocking with0.5% TWEEN®-20 (in PBS), biotinylated and non-biotinylated THIOMAB™antibody-phage (2×10¹⁰ phage particles) are incubated for 1 hour at roomtemperature; (3) the incubation with the phage is followed by incubationwith HRP labeled secondary antibody (anti-M13 phage coat protein, pVIIIprotein antibody); (4) standard HRP reactions are carried out and theabsorbance is measured at 450 nm; (5) thiol reactivity is measured bycalculating the ratio between OD₄₅₀ for streptavidin/OD₄₅₀ for thetarget protein (e.g., VEGF) such that a thiol reactivity value of 1indicates complete biotinylation of the cysteine thiol.

DNA encoding the cysteine engineered antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells,HEK293T cells, or other mammalian host cells, such as myeloma cells(U.S. Pat. No. 5,807,715; US 2005/0048572; US 2004/0229310) that do nototherwise produce the antibody protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. In most cases, theyields of the cysteine engineered antibodies are similar to wild typeantibodies.

After design and selection, cysteine engineered antibodies, e.g.,THIOMAB™ antibodies, with highly reactive unpaired Cys residues, may beproduced by: (i) expression in a bacterial, e.g., E. coli, system or amammalian cell culture system (WO 01/00245), e.g., Chinese Hamster Ovarycells (CHO) or HEK293 cells (e.g., HEK293T cells); and (ii) purificationusing common protein purification techniques (e.g., Lowman et al (1991)J. Biol. Chem. 266(17):10982-10988). In specific embodiments, theTHIOMAB™ antibodies are expressed in a mammalian cell expression system.In specific embodiments, the mammalian cell expression system is HEK293Tcells.

The structure positions of the engineered Cys residues of the heavy andlight chains can be numbered according to a sequential numbering system.This sequential numbering system is correlated to the Kabat numberingsystem (Kabat et al., (1991) Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md.) for the 4D5 antibody in FIGS. 13A and 13B. Using theKabat numbering system, the actual linear amino acid sequence of the maycontain fewer or additional amino acids corresponding to a shorteningof, or insertion into, a FR or CDR of the variable domain. Cysteineengineered heavy chain variant sites and light chain variant sites areidentified by the sequential numbering and Kabat numbering in FIGS. 13Aand 13B.

Thiol reactivity may also be generalized to certain domains of anantibody, such as the light chain constant domain (CL) and heavy chainconstant domains, CH1, CH2 and CH3. Cysteine replacements resulting inthiol reactivity values of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, and 0.95 and higher may be made in the heavy chain constant domainsα, δ, ε, γ, and μ of intact antibodies: IgA, IgD, IgE, IgG, and IgM,respectively, including the IgG subclasses: IgG1, IgG2, IgG3, IgG4, IgA,and IgA2.

Cysteine engineered antibodies may be generated as described, e.g., inU.S. Pat. No. 7,521,541 or International Patent Publication No. WO2006/034488, which are incorporated by reference herein in theirentirety. In some embodiments, the cysteine engineered antibody variantis a cysteine engineered antibody variant described in U.S. Pat. No.7,521,541 or International Patent Publication No. WO 2006/034488. Insome instances, the cysteine engineered antibody variant is a cysteineengineered antibody variant described in International PatentApplication Publication No. WO 2011/156328 or U.S. Pat. No. 9,000,130,which are incorporated by reference herein in their entirety. In someembodiments, the cysteine engineered antibody variant is a cysteineengineered antibody variant described in International PatentApplication Publication No. WO 2016/040856, which is incorporated hereinby reference in its entirety, for example, in Tables 1-4 of WO2016/040856.

For example, in certain embodiments, the cysteine mutation is selectedfrom the group consisting of HC-I195C, HC-S420C, HC-Y432C, and LC-G64C(according to Kabat numbering). In certain embodiments, the cysteinemutation is selected from the group consisting of HC-Y432C and LC-G64C(according to Kabat numbering). In certain embodiments, the cysteinemutation is a heavy chain mutation and is selected from the groupconsisting of Y33C, G162C, V184C, I195C, S420C, Y432C, and Q434C(according to Kabat numbering). In certain embodiments, the cysteinemutation is a heavy chain mutation and is selected from the groupconsisting of R19C, E46C, T57C, Y59C, A60C, M100cC, W103C, G162C, I195C,V258C, S420C, H425C, and N430C (according to Kabat numbering). Incertain embodiments, the cysteine mutation is a heavy chain mutation andis selected from the group consisting of Y33C, G162C, V184C, and I195C(according to Kabat numbering). In certain embodiments, the cysteinemutation is a heavy chain mutation and is selected from the groupconsisting of R19C, E46C, Y59C, A60C, M100cC, W103C, V258C, H425C, andN430C (according to Kabat numbering).

In certain embodiments, the cysteine mutation is a light chain mutationand is selected from the group consisting of Y55C, G64C, T85C, T180C,and N430C (according to Kabat numbering). In certain embodiments, thecysteine mutation is a light chain mutation and is selected from thegroup consisting of T31C, S52C, G64C, R66C, A193C, and N430C (accordingto Kabat numbering). In certain embodiments, the cysteine mutation is alight chain mutation and is selected from the group consisting of G64C,T85C, T180C, and N430C (according to Kabat numbering). In certainembodiments, the cysteine mutation is a light chain mutation and isselected from the group consisting of S52C, G64C, R66C, A193C, and N430C(according to Kabat numbering). In specific embodiments, the cysteinemutation in the light chain is selected from the group of cysteinemutations comprising LC-I106C, LC-R108C, LC-R142C, and LC-K149C(according to Kabat numbering) (see FIG. 13A; Table 2). In a particularembodiment, the cysteine mutation in the light chain is LC-K149C(according to Kabat numbering) (see FIG. 13A). In a particularembodiment, the cysteine mutation in the in the light chain is LC-V205C(according to Kabat numbering).

TABLE 2 Exemplary Light Chain Cysteine Mutations Sequence SEQ EU KabatResiude (+/− 5 Residues) ID NO. Numbering Numbering I GTKVE C KRTVA 70106 106 R KVEIK C TVAAP 71 108 108 R NNFYP C EAKVQ 72 142 142 K AKVQW CVDNAL 73 149 149

In particular embodiments, the cysteine mutation in the heavy chain isselected from the group of cysteine mutations consisting of HC-T114C,HC-A140C, HC-L174C, HC-L179C, HC-T187C, HC-T209C, HC-V262C, HC-G371C,HC-Y373C, HC-E382C, HC-S424C, HC-N434C, and HC-Q438C (according to EUnumbering) (see FIG. 13B; Table 3). In a particular embodiment, thecysteine mutation in the heavy chain is HC-A143C according to Kabatnumbering (i.e., HC-A140C according to EU numbering) (see FIG. 13B;Table 3). In a particular embodiment, the cysteine mutation in the heavychain is HC-A174C according to EU numbering (see FIG. 13B; Table 3). Ina particular embodiment, the cysteine mutation in the heavy chain isHC-A118C according to EU numbering (i.e., HC-A114C according to Kabatnumbering).

In a particular embodiment, any cysteine engineered antibody asdescribed herein has one of the following cysteine mutations: LC-K149Caccording to Kabat numbering and HC-A140C according to EU numbering (seeTables 2 and 3 and FIGS. 13A and 13B).

TABLE 3 Exemplary Heavy Chain Cysteine Mutations SEQ Sequence ID EUKabat Residue (+/-5 Residues) NO. Numbering Numbering T QGTLV C VSSAS 74114 110 A TSGGT C ALGCL 75 140 136 L TFPAV C QSSGL 76 174 170 L LQSSG CYSLSS 77 179 175 T LSSVV C VPSSS 78 187 183 T HKPSN C KVDKK 79 209 205 VPEVTC C VVDVS 80 262 258 G TCLVK C FYPSD 81 371 367 Y LVKGF C PSDIA 82373 369 E IAVEW C SNGQP 83 382 378 S QGNVF C CSVMH 84 424 420 N HEALH CHYTQK 85 434 430 Q HNHYT C KSLSL 86 438 434

In certain embodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of theheavy chain Fc region.

In certain embodiments, the cysteine engineered antibody may include oneor more heavy chain cysteine mutations selected from the groupconsisting of V2C, L4C, V5C, L110, R19C, F27C, I29C, T32C, Y33C, Q39C,A40C, K43C, L45C, E46C, T53C, G55C, T57C, R58C, Y59C, A60C, T68C, N76C,Y79C, Q81C, W95C, G96C, D101C, W103C, T116C, K117C, T135C, N155C, A158C,G162C, G174C, L175C, T183C, V184C, I195C, N199C, S203C, F239C, M248C,E254C, V258C, N272C, V278C, L305C, T331C, S333C, R340C, Q343C, K356C,E384C, S399C, K410C, Q414C, G416C, N417C, Y432C, T433C, K435C, S438C,L439C, M100cC, and N82aC (according to Kabat numbering). See also Table3 of International Patent Application No. WO 2016/040856.

In certain embodiments, the cysteine engineered antibody may include oneor more light chain cysteine mutations selected from the groupconsisting of S12C, S14C, G16C, R18C, T22C, R24C, Q27C, T31C, A32C,Q38C, K39C, G41C, K42C, P44C, Y49C, S50C, S52C, F53C, L54C, Y55C, S63C,G64C, R66C, D70C, T72C, T74C, S76C, Q79C, T85C, H91C, Y92C, P95C, T97C,F98C, K103C, E105C, K107C, P119C, K126C, T129C, S131C, Q147C, W148C,A153C, Q155C, S156C, S159C, Q160C, S162C, Q166C, T172C, T180C, V191C,A193C, E195C, V205C, T206C, and N210C (according to Kabat numbering).See also Table 4 of International Patent Application No. WO 2016/040856.

In certain embodiments, the cysteine engineered antibody may include oneor more cysteine mutations selected from the group consisting ofHC-I195C, HC-S420C, HC-Y432C, and LC-G640 (according to Kabatnumbering). See also Table 5 of International Patent Application No. WO2016/040856.

In certain embodiments, the cysteine engineered antibody includes alight chain cysteine mutation selected from the group of sitesconsisting of LC-T220, LC-K390, LC-Y490, LC-Y550, LC-T850, LC-T970,LC-I106C, LC-R108C, LC-R142C, LC-K149C, and LC-V205C (according to Kabatnumbering).

The cysteine engineered antibody may include any suitable number ofengineered cysteine residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more engineered cysteine residues. In someembodiments, the cysteine engineered antibody may include from 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 engineered cysteine residues. In someembodiments, the cysteine engineered antibody may include 1 engineeredcysteine residue. In some embodiments, the cysteine engineered antibodymay include 2 engineered cysteine residues. In some embodiments, thecysteine engineered antibody may include 3 engineered cysteine residues.

In any of the preceding embodiments, the cysteine engineered antibodymay include an engineered cysteine at the equivalent position as any ofthe cysteine mutations described above. For instance, if an antibodyincludes a native serine at position 118 (EU numbering), the serine canbe mutated to a cysteine to form an S118C mutation.

For example, the invention provides a cysteine engineered anti-VEGFantibody comprising a cysteine mutation in the heavy chain selected fromthe group consisting of HC-A118C, HC-A140C, and HC-L174C (EU numbering),or a cysteine mutation in the light chain selected from the groupconsisting of LC-V205C and LC-K149C (Kabat numbering), wherein theanti-VEGF antibody is any anti-VEGF antibody described herein, forexample, any anti-VEGF antibody described in Tables 8-10. In someembodiments, the anti-VEGF antibody is N94A.F83A.N82aR.Y58R (G6.31AARR). In some embodiments, the anti-VEGF antibody is G6.31 WT. In someembodiments, the anti-VEGF antibody is LC-N94A. In some embodiments, theanti-VEGF antibody is LC-N94A.LC-F83A. In some embodiments, theanti-VEGF antibody is LC-N94A.LC-F83A. In some embodiments, theanti-VEGF antibody is HC-A40E.HC-T57E (G6.31 AAEE). In some embodiments,the anti-VEGF antibody is HCcombo. In some embodiments, the anti-VEGFantibody is HCLC2. In some embodiments, the anti-VEGF antibody is HCLC4.In some embodiments, the anti-VEGF antibody is HCLC5. In someembodiments, the anti-VEGF antibody is HCLC3. In some embodiments, theanti-VEGF antibody is HCLC1. In some embodiments, the anti-VEGF antibodyis R19HCcombo. In some embodiments, the anti-VEGF antibody is R19HCLC2.In some embodiments, the anti-VEGF antibody is R19HCLC4. In someembodiments, the anti-VEGF antibody is R19HCLC5.

In a particular example, the invention provides a cysteine engineeredanti-VEGF antibody comprising an cysteine mutation in the heavy chainselected from the group consisting of HC-A1180, HC-A140C, and HC-L174C(EU numbering), or an cysteine mutation in the light chain selected fromthe group consisting of LC-V205C and LC-K149C (Kabat numbering), whereinthe antibody comprises the following six HVRs: (a) an HVR-H1 comprisingthe amino acid sequence of DYWIH (SEQ ID NO: 1); (b) an HVR-H2comprising the amino acid sequence of GITPAGGYTRYADSVKG (SEQ ID NO: 7);(c) an HVR-H3 comprising the amino acid sequence of FVFFLPYAMDY (SEQ IDNO: 3); (d) an HVR-L1 comprising the amino acid sequence of RASQDVSTAVA(SEQ ID NO: 8); (e) an HVR-L2 comprising the amino acid sequence ofSASFLYS (SEQ ID NO: 9); and (f) an HVR-L3 comprising the amino acidsequence of QQGYGAPFT (SEQ ID NO: 10). In some instances, the cysteineengineered anti-VEGF antibody includes the following four heavy chainvariable domain FRs: (a) an FR-H1 comprising the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTIS (SEQ ID NO: 13); (b) an FR-H2 comprisingthe amino acid sequence of WVRQAPGKGLEWVA (SEQ ID NO: 14); (c) an FR-H3comprising the amino acid sequence of RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR(SEQ ID NO: 15); and (d) an FR-H4 comprising the amino acid sequence ofWGQGTLVTVSS (SEQ ID NO: 16). In further instances, the cysteineengineered anti-VEGF antibody includes the following four light chainvariable domain FRs: (a) an FR-L1 comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 17); (b) an FR-L2 comprising theamino acid sequence of WYQQKPGKAPKLLIY (SEQ ID NO: 18); (c) an FR-L3comprising the amino acid sequence of GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC(SEQ ID NO: 19); and (d) an FR-L4 comprising the amino acid sequence ofFGQGTKVEIK (SEQ ID NO: 20). In some instances, the cysteine engineeredanti-VEGF antibody includes (a) a VH domain comprising an amino acidsequence of SEQ ID NO: 11 and (b) a VL domain comprising an amino acidsequence of SEQ ID NO: 12. In some instances, the parent antibody isG6.31 AARR. In some embodiments, the cysteine mutation is HC-A1180. Inother embodiments, the cysteine mutation is HC-A140C. In yet otherembodiments, the cysteine mutation is HC-L174C (EU numbering). In otherembodiments, the cysteine mutation is LC-V205C (Kabat numbering). Inother embodiments, the cysteine mutation is LC-K1490 (Kabat numbering).

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 90 and/or(b) a light chain comprising the amino acid sequence of SEQ ID NO: 89.

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 92 and/or(b) a light chain comprising the amino acid sequence of SEQ ID NO: 91.

In some instances, the invention provides an antibody comprising (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 94 and/or(b) a light chain comprising the amino acid sequence of SEQ ID NO: 93.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,and the like. Additional antibody conjugates are described herein, forexample, in Section G below and in Examples 1 and 2.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

f) Isoelectric Point Variants

The invention provides antibodies variants with altered isoelectricpoints. For example, the invention provides antibodies variants with areduced isoelectric point (pI), for example, as compared to an anti-VEGFantibody, for instance, G6.31. In some instances, the surface charge isreduced at physiological pH. In some instances, the anti-VEGF antibodyhas a pI equal to or lower than about 8 (e.g., about 8, about 7, about6, about 5, or about 4). In some instances, the antibody has a pI fromabout 4 to about 8 (e.g., about 4, about 5, about 6, about 7, or about8). In some instances, the anti-VEGF antibody has a pI from about 5 toabout 7 (e.g., about 5, about 6, or about 7). In some instances, theanti-VEGF antibody has a pI from about 5 to about 6 (e.g., about 5.1,about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about5.8, about 5.9, or about 6).

Antibodies of the invention may be engineered to have a reduced pI, forexample, by substituting wild-type amino acid residues at a givenposition with an amino acid having a lower pI. The pI of an amino acidcan be determined based on the pKa values of the amine (—NH₂),carboxylic acid (—COOH), and side-chain of the amino acid, which areknown in the art. In some embodiments, surface-exposed amino acidresidues may be substituted to reduce the pI of an antibody. In oneembodiment, surface-exposed amino acid residues may be substituted withglutamate (E). In one embodiment, surface-exposed amino acid residuesmay be substituted with aspartate (D).

B. Recombinant Methods and Compositions

Any of the antibodies (e.g., anti-VEGF antibodies, including cysteineengineered anti-VEGF antibodies) described herein may be produced usingrecombinant methods and compositions, for example, as described in U.S.Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encodingan anti-VEGF antibody described herein is provided. Such a nucleic acidmay encode an amino acid sequence comprising the VL and/or an amino acidsequence comprising the VH of the antibody (e.g., the light and/or heavychains of the antibody). In a further embodiment, one or more vectors(e.g., expression vectors) comprising such a nucleic acid are provided.In a further embodiment, a host cell comprising such a nucleic acid isprovided. In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one embodiment, the host cell is eukaryotic, for example, a ChineseHamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). Inone embodiment, a method of making an anti-VEGF antibody is provided,wherein the method comprises culturing a host cell comprising a nucleicacid encoding the antibody, as provided above, under conditions suitablefor expression of the antibody, and optionally recovering the antibodyfrom the host cell (or host cell culture medium).

For recombinant production of an antibody (e.g., an anti-VEGF antibody,including a cysteine engineered anti-VEGF antibody), nucleic acidencoding an antibody, for example, as described above, is isolated andinserted into one or more vectors for further cloning and/or expressionin a host cell. Such nucleic acid may be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, for example, U.S.Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. See also Charlton,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J., 2003), pp. 245-254, describing expression of antibodyfragments in E. coli. After expression, the antibody may be isolatedfrom the bacterial cell paste in a soluble fraction and can be furtherpurified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, for example,U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, for example, Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003).

C. Assays

Antibodies (e.g., anti-VEGF antibodies described herein, includingcysteine engineered anti-VEGF antibodies), as well as antibodyconjugates (e.g., antibody conjugates that include anti-VEGF antibodies(e.g., any anti-VEGF antibody provided herein)), may be identified,screened for, or characterized for their physical/chemical propertiesand/or biological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody (e.g., an anti-VEGF antibody, including acysteine engineered anti-VEGF antibody), or an antibody conjugatethereof, is tested for its antigen binding activity, e.g., by knownmethods such as ELISA, Western blot, etc.

In another aspect, competition assays may be used to identify anantibody that competes with an antibody as described herein, or anantibody conjugate thereof, for binding to an antigen (e.g., VEGF). Incertain embodiments, such a competing antibody binds to the same epitope(e.g., a linear or a conformational epitope) that is bound by anantibody as described herein. Detailed exemplary methods for mapping anepitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized VEGF is incubated in asolution comprising a first labeled antibody that binds to VEGF and asecond unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to VEGF. The second antibodymay be present in a hybridoma supernatant. As a control, immobilizedVEGF is incubated in a solution comprising the first labeled antibodybut not the second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to VEGF, excess unboundantibody is removed, and the amount of label associated with immobilizedVEGF is measured. If the amount of label associated with immobilizedVEGF is substantially reduced in the test sample relative to the controlsample, then that indicates that the second antibody is competing withthe first antibody for binding to VEGF. Similar assays may be performedfor other antigens. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying antibodies (e.g.,anti-VEGF antibodies, including cysteine engineered anti-VEGFantibodies), or antibody conjugates thereof, having biological activity.Biological activity may include, for example, binding to an antigen(e.g., VEGF (e.g., VEGF in the blood stream)), or a peptide fragmentthereof, either in vivo, in vitro, or ex vivo. In certain embodiments,biological activity may include blocking or neutralizing an antigen. Forexample, in certain embodiments, biological activity may includeblocking or neutralizing VEGF, or preventing VEGF from binding to aligand, for example, a receptor such as KDR or Flt-1. Antibodies, orantibody conjugates thereof, having such biological activity in vivoand/or in vitro are also provided. In certain embodiments, an antibodyof the invention, or an antibody conjugate thereof, is tested for suchbiological activity.

3. Stability Assays

In one aspect, assays are provided for determining the stability (e.g.,thermostability) of an antibody (e.g., an anti-VEGF antibody, includinga cysteine engineered anti-VEGF antibody), or an antibody conjugatethereof. For example, the stability of an antibody, or an antibodyconjugate thereof, may be determined using any method known in the art,for example, differential scanning fluorimetry (DSF), circular dichroism(CD), intrinsic protein fluorescence, differential scanning calorimetry,spectroscopy, light scattering (e.g., dynamic light scattering (DLS) andstatic light scattering (SLS), self-interaction chromatography (SIC).The stability of an antibody, or an antibody conjugate thereof, may bedetermined as described herein, for example, using DSF as described, forexample, in Examples 1 and 2 of International Patent Application No.PCT/US2016/053454. In some instances, the stability of an antibodyconjugate can be determined by size exclusion chromatography in-linewith refractive index and multi-angle light scattering detectors(SEC-RI-MALS), for example, as described in Example 1.

D. Pharmaceutical Formulations

Pharmaceutical formulations of an antibody (e.g., an anti-VEGF antibody,including a cysteine engineered anti-VEGF antibody) or antibodyconjugate thereof provided herein are prepared by mixing such antibodyor antibody conjugate having the desired degree of purity with one ormore optional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further include interstitialdrug dispersion agents such as soluble neutral-active hyaluronidaseglycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidaseglycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs and methods of use, including rHuPH20, aredescribed in US Patent Publication Nos. 2005/0260186 and 2006/0104968.In one aspect, a sHASEGP is combined with one or more additionalglycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, for example, byfiltration through sterile filtration membranes.

In certain embodiments, the pharmaceutical formulation includes one ormore additional compounds. In certain embodiments, the additionalcompound binds to a second biological molecule selected from the groupconsisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R; PDGF; angiopoietin;angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin;apelin/APJ; erythropoietin; complement factor D; TNFα; HtrA1; a VEGFreceptor; ST-2 receptor; and proteins genetically linked to age-relatedmacular degeneration (AMD) risk, such as complement pathway componentsC2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2;TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; andTNFRSF10A. In certain embodiments, the additional compound is anantibody or antigen-binding fragment thereof.

For example, in some instances, the additional compound is a bispecificantibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody, such asRG-7716 or any bispecific anti-VEGF/anti-Ang2 bispecific antibodydisclosed in WO 2010/069532 or WO 2016/073157.

In another example, in some instances, the additional compound is ananti-IL-6 antibody, for example, EBI-031 (Eleven Biotherapeutics; see,e.g., WO 2016/073890), siltuximab (SYLVANT®), olokizumab, clazakizumab,sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921,or a variant thereof.

In a still further example, in some instances, the additional compoundis an anti-IL-6R antibody, for example, tocilizumab (ACTEMRA®) (see,e.g., WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or avariant thereof.

E. Therapeutic Methods and Compositions

Any of the antibodies (e.g., anti-VEGF antibodies, including cysteineengineered anti-VEGF antibodies) or antibody conjugates thereof (e.g.,monodisperse HA conjugates) provided herein may be used in therapeuticmethods.

In one aspect, an anti-VEGF antibody (e.g., an engineered cysteineanti-VEGF antibody) for use as a medicament is provided. In anotheraspect, an antibody conjugate (e.g., a monodisperse HA conjugate) foruse as a medicament is provided. In further aspects, the inventionprovides an anti-VEGF antibody (e.g., an engineered cysteine anti-VEGFantibody) for use in treating a disorder associated with pathologicalangiogenesis. In another aspect, the invention provides an antibodyconjugate (e.g., a monodisperse HA conjugate) for use in treating adisorder associated with pathological angiogenesis. In some embodiments,the disorder associated with pathological angiogenesis is an oculardisorder. In some instances, the ocular disorder is AMD (e.g., wet AMD,dry AMD, intermediate AMD, advanced AMD, or geographic atrophy (GA)),macular degeneration, macular edema, DME (e.g., focal, non-center DME ordiffuse, center-involved DME), retinopathy, diabetic retinopathy (DR)(e.g., proliferative DR (PDR), non-proliferative DR (NPDR), orhigh-altitude DR), other ischemia-related retinopathies, ROP, retinalvein occlusion (RVO) (e.g., central (CRVO) and branched (BRVO) forms),CNV (e.g., myopic CNV), corneal neovascularization, diseases associatedwith corneal neovascularization, retinal neovascularization, diseasesassociated with retinal/choroidal neovascularization, pathologic myopia,von Hippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats'disease, Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis,ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa(RP), hypertensive retinopathy, retinal angiomatous proliferation,macular telangiectasia, iris neovascularization, intraocularneovascularization, retinal degeneration, cystoid macular edema (CME),vasculitis, papilloedema, retinitis, conjunctivitis (e.g., infectiousconjunctivitis and non-infectious (e.g., allergic) conjunctivitis),Leber congenital amaurosis, uveitis (including infectious andnon-infectious uveitis), choroiditis (e.g., multifocal choroiditis),ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, orSjögren's disease.

In another aspect, an anti-VEGF antibody (e.g., an engineered cysteineanti-VEGF antibody) for use in a method of treatment is provided. Inanother aspect, an antibody conjugate (e.g., a monodisperse HAconjugate) for use in a method of treatment is provided. In certaininstances, the invention provides an anti-VEGF antibody (e.g., anengineered cysteine anti-VEGF antibody) for use in a method of treatinga subject having a disorder associated with pathological angiogenesiscomprising administering to the individual an effective amount of theanti-VEGF antibody. The invention also provides an antibody conjugate(e.g., a monodisperse HA conjugate) for use in a method of treating asubject having a disorder associated with pathological angiogenesiscomprising administering to the individual an effective amount of theantibody conjugate. In some instances, the disorder associated withpathological angiogenesis is an ocular disorder. In some instances, theocular disorder is AMD (e.g., wet AMD, dry AMD, intermediate AMD,advanced AMD, or geographic atrophy (GA)), macular degeneration, macularedema, DME (e.g., focal, non-center DME or diffuse, center-involvedDME), retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR(PDR), non-proliferative DR (NPDR), or high-altitude DR), otherischemia-related retinopathies, ROP, retinal vein occlusion (RVO) (e.g.,central (CRVO) and branched (BRVO) forms), CNV (e.g., myopic CNV),corneal neovascularization, diseases associated with cornealneovascularization, retinal neovascularization, diseases associated withretinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats' disease,Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis, ocularneovascular disease, neovascular glaucoma, retinitis pigmentosa (RP),hypertensive retinopathy, retinal angiomatous proliferation, maculartelangiectasia, iris neovascularization, intraocular neovascularization,retinal degeneration, cystoid macular edema (CME), vasculitis,papilloedema, retinitis, conjunctivitis (e.g., infectious conjunctivitisand non-infectious (e.g., allergic) conjunctivitis), Leber congenitalamaurosis, uveitis (including infectious and non-infectious uveitis),choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis,blepharitis, dry eye, traumatic eye injury, or Sjögren's disease.

In some instances, the invention provides an anti-VEGF antibody (e.g.,an engineered cysteine anti-VEGF antibody) for use in reducing orinhibiting angiogenesis in a subject. In another aspect, an antibodyconjugate (e.g., a monodisperse HA conjugate) for use in reducing orinhibiting angiogenesis in a subject is provided. In certainembodiments, the invention provides an anti-VEGF antibody (e.g., anengineered cysteine anti-VEGF antibody) for use in a method of reducingor inhibiting angiogenesis in a subject comprising administering to theindividual an effective of the anti-VEGF antibody to reduce or inhibitangiogenesis. The invention also provides an antibody conjugate (e.g., amonodisperse HA conjugate) for use in a method of reducing or inhibitingangiogenesis in a subject comprising administering to the individual aneffective amount of the antibody conjugate. A “subject” according to anyof the above uses may be a human.

The invention provides for the use of an anti-VEGF antibody (e.g., anengineered cysteine anti-VEGF antibody) in the manufacture orpreparation of a medicament. The invention also provides for the use ofan antibody conjugate (e.g., a monodisperse HA conjugate) in themanufacture or preparation of a medicament. For example, in oneinstance, the medicament is for treatment of a disorder associated withpathological angiogenesis. In a further instance, the medicament is foruse in a method of treating a disorder associated with pathologicalangiogenesis comprising administering to a subject having a disorderassociated with pathological angiogenesis an effective amount of themedicament. In some instances, the disorder associated with pathologicalangiogenesis is an ocular disorder. In some instances, the oculardisorder is AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD,or geographic atrophy (GA)), macular degeneration, macular edema, DME(e.g., focal, non-center DME or diffuse, center-involved DME),retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR (PDR),non-proliferative DR (NPDR), or high-altitude DR), otherischemia-related retinopathies, ROP, retinal vein occlusion (RVO) (e.g.,central (CRVO) and branched (BRVO) forms), CNV (e.g., myopic CNV),corneal neovascularization, diseases associated with cornealneovascularization, retinal neovascularization, diseases associated withretinal/choroidal neovascularization, pathologic myopia, vonHippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats' disease,Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis, ocularneovascular disease, neovascular glaucoma, retinitis pigmentosa (RP),hypertensive retinopathy, retinal angiomatous proliferation, maculartelangiectasia, iris neovascularization, intraocular neovascularization,retinal degeneration, cystoid macular edema (CME), vasculitis,papilloedema, retinitis, conjunctivitis (e.g., infectious conjunctivitisand non-infectious (e.g., allergic) conjunctivitis), Leber congenitalamaurosis, uveitis (including infectious and non-infectious uveitis),choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis,blepharitis, dry eye, traumatic eye injury, or Sjögren's disease. In afurther instance, the medicament is for reducing or inhibitingangiogenesis in a subject. In a further instance, the medicament is foruse in a method of reducing or inhibiting angiogenesis in a subjectcomprising administering to the subject an amount effective of themedicament to reduce or inhibit angiogenesis. In any of the precedinguses of medicaments, the method may include administering to theindividual an effective amount of at least one additional therapeuticagent, e.g., as described below.

The invention provides a method for treating a disorder associated withpathological angiogenesis. In one embodiment, the method comprisesadministering to an individual having a disorder associated withpathological angiogenesis an effective amount of an anti-VEGF antibody(e.g., an engineered cysteine anti-VEGF antibody). In another example,the method comprises administering to an individual having a disorderassociated with pathological angiogenesis an effective amount of anantibody conjugate (e.g., a monodisperse HA conjugate). In someinstances, the disorder associated with pathological angiogenesis is anocular disorder. In some instances, the ocular disorder is AMD (e.g.,wet AMD, dry AMD, intermediate AMD, advanced AMD, or geographic atrophy(GA)), macular degeneration, macular edema, DME (e.g., focal, non-centerDME or diffuse, center-involved DME), retinopathy, diabetic retinopathy(DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), orhigh-altitude DR), other ischemia-related retinopathies, ROP, retinalvein occlusion (RVO) (e.g., central (CRVO) and branched (BRVO) forms),CNV (e.g., myopic CNV), corneal neovascularization, diseases associatedwith corneal neovascularization, retinal neovascularization, diseasesassociated with retinal/choroidal neovascularization, pathologic myopia,von Hippel-Lindau disease, histoplasmosis of the eye, FEVR, Coats'disease, Norrie Disease, OPPG, subconjunctival hemorrhage, rubeosis,ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa(RP), hypertensive retinopathy, retinal angiomatous proliferation,macular telangiectasia, iris neovascularization, intraocularneovascularization, retinal degeneration, cystoid macular edema (CME),vasculitis, papilloedema, retinitis, conjunctivitis (e.g., infectiousconjunctivitis and non-infectious (e.g., allergic) conjunctivitis),Leber congenital amaurosis, uveitis (including infectious andnon-infectious uveitis), choroiditis (e.g., multifocal choroiditis),ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, orSjögren's disease. In further instances, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, as described below. A “subject” accordingto any of the above methods may be a human.

It is contemplated that the antibody (e.g., cysteine engineeredanti-VEGF antibody) or antibody conjugate (e.g., monodisperse HAconjugate) of the present invention may be used to treat a mammal. Inone embodiment, the antibody (e.g., cysteine engineered anti-VEGFantibody) or antibody conjugate (e.g., monodisperse HA conjugate) isadministered to a nonhuman mammal for the purposes of obtainingpreclinical data, for example. Exemplary nonhuman mammals to be treatedinclude nonhuman primates, dogs, cats, rodents (e.g., mice and rats) andother mammals in which preclinical studies are performed. Such mammalsmay be established animal models for a disease to be treated with theantibody or may be used to study toxicity or pharmacokinetics of theantibody of interest. In each of these embodiments, dose escalationstudies may be performed in the mammal. The antibody (e.g., cysteineengineered anti-VEGF antibody) or antibody conjugate (e.g., monodisperseHA conjugate) may be administered to a host rodent in a solid tumormodel, for example. The antibody or antibody conjugate may beadministered to a host (e.g., a rodent, e.g., a rabbit) for ocularpharmacokinetic studies, for example, by intravitreal administration(e.g., intravitreal injection) or using a port delivery device.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the antibodies (e.g., cysteine engineered anti-VEGFantibodies) or antibody conjugates (e.g., monodisperse HA conjugates)provided herein, for example, for use in any of the above therapeuticmethods. In one embodiment, a pharmaceutical formulation comprises anyof the antibodies (e.g., cysteine engineered anti-VEGF antibodies) orantibody conjugates (e.g., monodisperse HA conjugates) provided hereinand a pharmaceutically acceptable carrier. In another embodiment, apharmaceutical formulation comprises any of the antibodies (e.g.,cysteine engineered anti-VEGF antibodies) or antibody conjugates (e.g.,monodisperse HA conjugates) provided herein and at least one additionaltherapeutic agent, for example, as described below. In certainembodiments, the pharmaceutical formulation comprises one or moreadditional compounds. In certain embodiments, the additional compoundbinds to a second biological molecule selected from the group consistingof IL-1β; IL-6; IL-6R; IL-13; IL-13R; PDGF; angiopoietin; Ang2; Tie2;S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ;erythropoietin; complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2receptor; and proteins genetically linked to age-related maculardegeneration (AMD) risk, such as complement pathway components C2,factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3;HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC, COL10A1; andTNFRSF10A. In certain embodiments, the additional compound is anantibody or antigen-binding fragment thereof. For example, in someinstances, the additional compound is a bispecific antibody (e.g., ananti-VEGF/anti-Ang2 bispecific antibody, such as RG-7716 or anybispecific anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO2010/069532 or WO 2016/073157 or a variant thereof. In another example,in some instances, the additional compound is an anti-IL-6 antibody, forexample, EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890),siltuximab (SYLVANT®), olokizumab, clazakizumab, sirukumab, elsilimomab,gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof. Ina still further example, in some instances, the additional compound isan anti-IL-6R antibody, for example, tocilizumab (ACTEMRA®) (see, e.g.,WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or avariant thereof.

Antibodies (e.g., cysteine engineered anti-VEGF antibodies) or antibodyconjugates (e.g., monodisperse HA conjugates) can be used either aloneor in combination with other agents in a therapy. For instance, anantibody (e.g., a cysteine engineered anti-VEGF antibody) or antibodyconjugate (e.g., a monodisperse HA conjugate) may be co-administeredwith at least one additional therapeutic agent. In certain embodiments,an additional therapeutic agent is another antibody, an anti-angiogenicagent, an immunosuppressive agent, a cytokine, a cytokine antagonist, acorticosteroid, an anti-emetic, a cancer vaccine, an analgesic, orcombinations thereof.

For example, in certain embodiments, any of the preceding methodsfurther comprises administering one or more additional compounds. Incertain embodiments, the antibody (e.g., a cysteine engineered anti-VEGFantibody) or antibody conjugate (e.g., a monodisperse HA conjugate) isadministered simultaneously with the additional compound(s). In certainembodiments, the antibody or antibody conjugate is administered beforeor after the additional compound(s). In certain embodiments, theadditional compound binds to a second biological molecule selected fromthe group consisting of IL-1β; IL-6; IL-6R; IL-13; IL-13R; PDGF;angiopoietin; Ang2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1;betacellulin; apelin/APJ; erythropoietin; complement factor D; TNFα;HtrA1; a VEGF receptor; ST-2 receptor; and proteins genetically linkedto AMD risk, such as complement pathway components C2, factor B, factorH, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8(IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. Incertain embodiments, the additional compound is an antibody orantigen-binding fragment thereof.

In certain embodiments according to (or as applied to) any of theembodiments above, the ocular disorder is an intraocular neovasculardisease selected from the group consisting of proliferativeretinopathies, choroidal neovascularization (CNV), age-related maculardegeneration (AMD), diabetic and other ischemia-related retinopathies,diabetic macular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, retinal vein occlusion (RVO), including CRVOand BRVO, corneal neovascularization, retinal neovascularization, andretinopathy of prematurity (ROP). For example, in some instances, theadditional compound is a bispecific antibody (e.g., ananti-VEGF/anti-Ang2 bispecific antibody, such as RG-7716 or anybispecific anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO2010/069532 or WO 2016/073157 or a variant thereof. In another example,in some instances, the additional compound is an anti-IL-6 antibody, forexample, EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890),siltuximab (SYLVANT®), olokizumab, clazakizumab, sirukumab, elsilimomab,gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof. Ina still further example, in some instances, the additional compound isan anti-IL-6R antibody, for example, tocilizumab (ACTEMRA®) (see, e.g.,WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or avariant thereof.

In some instances, an antibody (e.g., a cysteine engineered anti-VEGFantibody) or an antibody conjugate (e.g., a monodisperse HA conjugate)of the invention may be administered in combination with at least oneadditional therapeutic agent for treatment of an ocular disorder, forexample, an ocular disorder described herein (e.g., AMD (e.g., wet AMD),DME, DR, or RVO). Exemplary additional therapeutic agents forcombination therapy for treatment of ocular disorders include, withoutlimitation, anti-angiogenic agents, such as VEGF antagonists, including,for example, anti-VEGF antibodies (e.g., the anti-VEGF Fab LUCENTIS®(ranibizumab)), soluble receptor fusion proteins (e.g., the recombinantsoluble receptor fusion protein EYLEA® (aflibercept, also known as VEGFTrap Eye; Regeneron/Aventis)), aptamers (e.g., the anti-VEGF pegylatedaptamer MACUGEN® (pegaptanib sodium; NeXstar Pharmaceuticals/OSIPharmaceuticals)), and VEGFR tyrosine kinase inhibitors (e.g.,4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT®(sunitinib)); Tryptophanyl-tRNA synthetase (TrpRS); squalamine; RETAANE®(anecortave acetate for depot suspension; Alcon, Inc.); CombretastatinA4 Prodrug (CA4P); MIFEPREX® (mifepristone-ru486); subtenontriamcinolone acetonide; intravitreal crystalline triamcinoloneacetonide; matrix metalloproteinase inhibitors (e.g., Prinomastat(AG3340; Pfizer)); fluocinolone acetonide (including fluocinoloneintraocular implant; Bausch & Lomb/Control Delivery Systems); linomide;inhibitors of integrin β3 function; angiostatin, and combinationsthereof. These and other therapeutic agents that can be administered incombination with an antibody conjugate of the invention are described,for example, in U.S. Patent Application No. US 2014/0017244, which isincorporated herein by reference in its entirety.

Further examples of additional therapeutic agents that can be used incombination with an antibody (e.g., a cysteine engineered anti-VEGFantibody) or an antibody conjugate (e.g., a monodisperse HA conjugate)of the invention for treatment of an ocular disorder (e.g., AMD, DME,DR, or RVO), include, but are not limited to, VISUDYNE® (verteporfin; alight-activated drug that is typically used in conjunction withphotodynamic therapy with a non-thermal laser), PKC412, Endovion (NS3728; NeuroSearch A/S), neurotrophic factors (e.g., glial derivedneurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)),diltiazem, dorzolamide, PHOTOTROP®, 9-cis-retinal, eye medication (e.g.,phospholine iodide, echothiophate, or carbonic anhydrase inhibitors),veovastat (AE-941; AEterna Laboratories, Inc.), Sirna-027 (AGF-745; SimaTherapeutics, Inc.), neurotrophins (including, by way of example only,NT-4/5, Genentech), Cand5 (Acuity Pharmaceuticals), INS-37217 (InspirePharmaceuticals), integrin antagonists (including those from Jerini AGand Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E(BioDiem Ltd.), thalidomide (as used, for example, by EntreMed, Inc.),cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan/Oculex),DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate(University of Michigan), LYN-002 (Lynkeus Biotech), microalgal compound(Aquasearch/Albany, Mera Pharmaceuticals), D-9120 (Celltech Group plc),ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme/Celtrix), tyrosinekinase inhibitors (e.g., those from Allergan, SUGEN, or Pfizer),NX-278-L (NeXstar Pharmaceuticals/Gilead Sciences), Opt-24 (OPTIS FranceSA), retinal cell ganglion neuroprotectants (Cogent Neurosciences),N-nitropyrazole derivatives (Texas A&M University System), KP-102(Krenitsky Pharmaceuticals), cyclosporin A, therapeutic agents used inphotodynamic therapy (e.g., VISUDYNE®; receptor-targeted PDT,Bristol-Myers Squibb, Co.; porfimer sodium for injection with PDT;verteporfin, QLT Inc.; rostaporfin with PDT, Miravent MedicalTechnologies; talaporfin sodium with PDT, Nippon Petroleum; andmotexafin lutetium, Pharmacyclics, Inc.), antisense oligonucleotides(including, by way of example, products tested by Novagali Pharma SA andISIS-13650, Isis Pharmaceuticals), and combinations thereof.

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or antibodyconjugate (e.g., a monodisperse HA conjugate) of the invention may beadministered in combination with a therapy or surgical procedure fortreatment of an ocular disorder (e.g., AMD, DME, DR, or RVO), including,for example, laser photocoagulation (e.g., panretinal photocoagulation(PRP)), drusen lasering, macular hole surgery, macular translocationsurgery, implantable miniature telescopes, PHI-motion angiography (alsoknown as micro-laser therapy and feeder vessel treatment), proton beamtherapy, microstimulation therapy, retinal detachment and vitreoussurgery, scleral buckle, submacular surgery, transpupillarythermotherapy, photosystem I therapy, use of RNA interference (RNAi),extracorporeal rheopheresis (also known as membrane differentialfiltration and rheotherapy), microchip implantation, stem cell therapy,gene replacement therapy, ribozyme gene therapy (including gene therapyfor hypoxia response element, Oxford Biomedica; Lentipak, Genetix; andPDEF gene therapy, GenVec), photoreceptor/retinal cells transplantation(including transplantable retinal epithelial cells, Diacrin, Inc.;retinal cell transplant, Cell Genesys, Inc.), acupuncture, andcombinations thereof.

In some instances, an antibody (e.g., a cysteine engineered anti-VEGFantibody) or antibody conjugate (e.g., a monodisperse HA conjugate) ofthe invention can be administered in combination with an anti-angiogenicagent for treatment of an ocular disorder (e.g., AMD, DME, DR, or RVO).Any suitable anti-angiogenic agent can be used in combination with anantibody (e.g., a cysteine engineered anti-VEGF antibody) or an antibodyconjugate of the invention, including, but not limited to, those listedby Carmeliet et al. Nature 407:249-257, 2000. In some embodiments, theanti-angiogenic agent is a VEGF antagonist, including, but not limitedto, an anti-VEGF antibody (e.g., the anti-VEGF Fab LUCENTIS®(ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single-chainantibody fragment; Novartis), or a bispecific anti-VEGF antibody (e.g.,an anti-VEGF/anti-angiopoeitin 2 bispecific antibody such as RG-7716;Roche)), a soluble recombinant receptor fusion protein (e.g., EYLEA®(aflibercept)), a VEGF variant, a soluble VEGFR fragment, an aptamercapable of blocking VEGF (e.g., pegaptanib) or VEGFR, a neutralizinganti-VEGFR antibody, a small molecule inhibitor of VEGFR tyrosinekinases, an anti-VEGF DARPin® (e.g., abicipar pegol), a smallinterfering RNAs which inhibits expression of VEGF or VEGFR, a VEGFRtyrosine kinase inhibitor (e.g.,4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT®(sunitinib)), and combinations thereof. In some instances, thebispecific anti-VEGF antibody binds to a second biological molecule,including but not limited to IL-1β; IL-6; IL-6R; PDGF (e.g., PDGF-BB);angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1;betacellulin; apelin/APJ; erythropoietin; complement factor D; TNFα;HtrA1; a VEGF receptor (e.g., VEGFR1, VEGFR2, VEGFR3, mbVEGFR, orsVEGFR); ST-2 receptor; and proteins genetically linked to age-relatedmacular degeneration (AMD) risk, such as complement pathway componentsC2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2;TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; andTNFRSF10A. For example, in some instances, the additional compound is abispecific antibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody,such as RG-7716 or any bispecific anti-VEGF/anti-Ang2 bispecificantibody disclosed in WO 2010/069532 or WO 2016/073157 or a variantthereof.

Other suitable anti-angiogenic agents that may be administered incombination with an antibody (e.g., a cysteine engineered anti-VEGFantibody) or an antibody conjugate (e.g., a monodisperse HA conjugate)of the invention for treatment of an ocular disorder (e.g., AMD, DME,DR, or RVO) include corticosteroids, angiostatic steroids, anecortaveacetate, angiostatin, endostatin, tyrosine kinase inhibitors, matrixmetalloproteinase (MMP) inhibitors, insulin-like growth factor-bindingprotein 3 (IGFBP3), stromal derived factor (SDF-1) antagonists (e.g.,anti-SDF-1 antibodies), pigment epithelium-derived factor (PEDF),gamma-secretase, Delta-like ligand 4, integrin antagonists,hypoxia-inducible factor (HIF)-1α antagonists, protein kinase CK2antagonists, agents that inhibit stem cell (e.g., endothelial progenitorcell) homing to the site of neovascularization (e.g., an anti-vascularendothelial cadherin (CD-144) antibody and/or an anti-SDF-1 antibody),and combinations thereof.

In a further example, in some instances, an antibody (e.g., a cysteineengineered anti-VEGF antibody) or an antibody conjugate (e.g., amonodisperse HA conjugate) of the invention can be administered incombination with an agent that has activity against neovascularizationfor treatment of an ocular disorder (e.g., AMD, DME, DR, or RVO), suchas an anti-inflammatory drug, a mammalian target of rapamycin (mTOR)inhibitor (e.g., rapamycin, AFINITOR® (everolimus), and TORISEL®(temsirolimus)), cyclosporine, a tumor necrosis factor (TNF) antagonist(e.g., an anti-TNFα antibody or antigen-binding fragment thereof (e.g.,infliximab, adalimumab, certolizumab pegol, and golimumab) or a solublereceptor fusion protein (e.g., etanercept)), an anti-complement agent, anonsteroidal antiinflammatory agent (NSAID), or combinations thereof.

In a still further example, in some instances, an antibody (e.g., acysteine engineered anti-VEGF antibody) or an antibody conjugate (e.g.,a monodisperse HA conjugate) of the invention can be administered incombination with an agent that is neuroprotective and can potentiallyreduce the progression of dry AMD to wet AMD, such as the class of drugscalled the “neurosteroids,” which include drugs such asdehydroepiandrosterone (DHEA) (brand names: PRASTERA™ and FIDELIN®),dehydroepiandrosterone sulfate, and pregnenolone sulfate.

Any suitable AMD therapeutic agent can be administered as an additionaltherapeutic agent in combination with an antibody (e.g., a cysteineengineered anti-VEGF antibody) or an antibody conjugate (e.g., amonodisperse HA conjugate) of the invention for treatment of an oculardisorder (e.g., AMD, DME, DR, or RVO), including, but not limited to, aVEGF antagonist, for example, an anti-VEGF antibody (e.g., LUCENTIS®(ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single-chainantibody fragment; Novartis), or a bispecific anti-VEGF antibody (e.g.,an anti-VEGF/anti-angiopoeitin 2 bispecific antibody such as RG-7716;Roche)), a soluble VEGF receptor fusion protein (e.g., EYLEA®(aflibercept)), an anti-VEGF DARPin® (e.g., abicipar pegol; MolecularPartners AG/Allergan), or an anti-VEGF aptamer (e.g., MACUGEN®(pegaptanib sodium)); a platelet-derived growth factor (PDGF)antagonist, for example, an anti-PDGF antibody, an anti-PDGFR antibody(e.g., REGN2176-3), an anti-PDGF-BB pegylated aptamer (e.g., FOVISTA®;Ophthotech/Novartis), a soluble PDGFR receptor fusion protein, or a dualPDGF/VEGF antagonist (e.g., a small molecule inhibitor (e.g., DE-120(Santen) or X-82 (TyrogeneX)) or a bispecific anti-PDGF/anti-VEGFantibody)); VISUDYNE® (verteporfin) in combination with photodynamictherapy; an antioxidant; a complement system antagonist, for example, acomplement factor C5 antagonist (e.g., a small molecule inhibitor (e.g.,ARC-1905; Opthotech) or an anti-05 antibody (e.g., LFG-316; Novartis), aproperdin antagonist (e.g., an anti-properdin antibody, e.g., CLG-561;Alcon), or a complement factor D antagonist (e.g., an anti-complementfactor D antibody, e.g., lampalizumab; Roche)); a visual cycle modifier(e.g., emixustat hydrochloride); squalamine (e.g., OHR-102; OhrPharmaceutical); vitamin and mineral supplements (e.g., those describedin the Age-Related Eye Disease Study 1 (AREDS1; zinc and/orantioxidants) and Study 2 (AREDS2; zinc, antioxidants, lutein,zeaxanthin, and/or omega-3 fatty acids)); a cell-based therapy, forexample, NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC celltransplantation (StemCells), CNTO-2476 (Janssen), OpRegen (Cell CureNeurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics);a tissue factor antagonist (e.g., h1-con1; Iconic Therapeutics); analpha-adrenergic receptor agonist (e.g., brimonidine tartrate); apeptide vaccine (e.g., S-646240; Shionogi); an amyloid beta antagonist(e.g., an anti-beta amyloid monoclonal antibody, e.g., GSK-933776); anS1P antagonist (e.g., an anti-S1P antibody, e.g., iSONEP™; Lpath Inc); aROBO4 antagonist (e.g., an anti-ROBO4 antibody, e.g., DS-7080a; DaiichiSankyo); a lentiviral vector expressing endostatin and angiostatin(e.g., RetinoStat); and any combination thereof. In some instances, AMDtherapeutic agents (including any of the preceding AMD therapeuticagents) can be co-formulated. For example, the anti-PDGFR antibodyREGN2176-3 can be co-formulated with aflibercept (EYLEA®). In someinstances, such a co-formulation can be administered in combination withan antibody of the invention. In some instances, the ocular disorder isAMD (e.g., wet AMD).

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with LUCENTIS® (ranibizumab) fortreatment of an ocular disorder (e.g., AMD, DME, DR, or RVO). LUCENTIS®(ranibizumab) may be administered, for example, at 0.3 mg/eye or 0.5mg/eye by intravitreal injection, for example, every month. In someinstances, the ocular disorder is AMD (e.g., wet AMD).

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with EYLEA® (aflibercept) fortreatment of an ocular disorder (e.g., AMD, DME, DR, or RVO). EYLEA®(aflibercept) may be administered, for example, at 2 mg/eye byintravitreal injection, for example, every four weeks (Q4W), or Q4W forthe first three months, followed by injections once every two months formaintenance. In some instances, the ocular disorder is AMD (e.g., wetAMD).

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with MACUGEN® (pegaptanib sodium) fortreatment of an ocular disorder (e.g., AMD, DME, DR, or RVO). MACUGEN®(pegaptanib sodium) may be administered, for example, at 0.3 mg/eye byintravitreal injection every six weeks. In some instances, the oculardisorder is AMD (e.g., wet AMD).

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with VISUDYNE® (verteporfin) incombination with photodynamic therapy for treatment of an oculardisorder (e.g., AMD, DME, DR, or RVO). VISUDYNE® can be administered,for example, by intravenous infusion at any suitable dose (e.g., 6 mg/m²of body surface area) and delivered once every three months (e.g., over10 minutes of infusion). In some instances, the ocular disorder is AMD(e.g., wet AMD).

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with a PDGF antagonist for treatmentof an ocular disorder (e.g., AMD, DME, DR, or RVO). Exemplary PDGFantagonists which may be used in combination with an antibody of theinvention include an anti-PDGF antibody, an anti-PDGFR antibody, a smallmolecule inhibitor (e.g., squalamine), an anti-PDGF-B pegylated aptamersuch as FOVISTA® (E10030; Ophthotech/Novartis), or a dual PDGF/VEGFantagonist (e.g., a small molecule inhibitor (e.g., DE-120 (Santen) orX-82 (TyrogeneX)) or a bispecific anti-PDGF/anti-VEGF antibody). Forexample, FOVISTA® can be administered as an adjunct therapy to anantibody of the invention. FOVISTA® can be administered at any suitabledose, for example, from 0.1 mg/eye to 2.5 mg/eye, e.g., at 0.3 mg/eye or1.5 mg/eye, for example, by intravitreal injection, for example everyfour weeks (04W). OHR-102 (squalamine lactate ophalmic solution, 0.2%)can be administered by eye drop, for example, twice daily. OHR-102 canbe administered in combination with VEGF antagonists such as LUCENTIS®or EYLEA®. In some embodiments, an antibody conjugate of the inventioncan be administered in combination with OHR-102, LUCENTIS®, and/orEYLEA®. In some instances, the ocular disorder is AMD (e.g., wet AMD).

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with RTH-258 for treatment of anocular disorder (e.g., AMD, DME, DR, or RVO). RTH-258 can beadministered, for example, by intravitreal injection or eye infusion.For intravitreal injection, RTH-258 can be administered at any suitabledose (e.g., 3 mg/eye or 6 mg/eye), for example, once every four weeks(04W) for the first three months as loading, followed by injection every12 weeks (Q12W) or every eight weeks (Q8W) for maintenance. In someinstances, the ocular disorder is AMD (e.g., wet AMD).

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with abicipar pegol for treatment ofan ocular disorder (e.g., AMD, DME, DR, or RVO). Abicipar pegol can beadministered, for example, by intravitreal injection. Abicipar pegol canbe administered at any suitable dose (e.g., 1 mg/eye, 2 mg/eye, 3mg/eye, 4 mg/eye, or 4.2 mg/eye), for example, once every four weeks(04W) for the first three months as loading, followed by injection every12 weeks (Q12W) or every eight weeks (Q8W) for maintenance. In someinstances, the ocular disorder is AMD (e.g., wet AMD).

Any suitable DME and/or DR therapeutic agent can be administered incombination with an antibody (e.g., a cysteine engineered anti-VEGFantibody) an antibody conjugate (e.g., a monodisperse HA conjugate) ofthe invention for treatment of an ocular disorder (e.g., AMD, DME, DR,or RVO), including, but not limited, to a VEGF antagonist (e.g.,LUCENTIS® or EYLEA®), a corticosteroid (e.g., a corticosteroid implant(e.g., OZURDEX® (dexamethasone intravitreal implant) or ILUVIEN®(fluocinolone acetonide intravitreal implant)) or a corticosteroidformulated for administration by intravitreal injection (e.g.,triamcinolone acetonide)), or combinations thereof. In some instances,the ocular disorder is DME and/or DR.

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with LUCENTIS® (ranibizumab) fortreatment of DME and/or DR (e.g., NPDR or PDR). LUCENTIS® (ranibizumab)may be administered, for example, at 0.3 mg/eye or 0.5 mg/eye byintravitreal injection, for example, every four weeks (04W).

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with EYLEA® (aflibercept) fortreatment of DME and/or DR (e.g., NPDR or PDR). EYLEA® (aflibercept) maybe administered, for example, at 2 mg/eye by intravitreal injection, forexample, every four weeks (Q4W), or Q4W for the first five months,followed by injections once every eight weeks (Q8W) for maintenance.

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with OZURDEX® (dexamethasoneintravitreal implant) for treatment of DME and/or DR. OZURDEX® can beadministered as a 0.7 mg dexamethasone intravitreal implant, which canbe administered up to every six months.

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the inventioncan be administered in combination with ILUVIEN® (dexamethasoneintravitreal implant) for treatment of DME and/or DR. OZURDEX® can beadministered as a 0.19 mg fluocinolone acetonide intravitreal implant,which can be eluted at a rate of 0.25 μg/day, and can last up to about36 months.

In some cases, the TAO/PRN treatment regimen or TAE treatment regimenmay be used to administer an AMD therapeutic agent (e.g., ranibizumab oraflibercept) in combination with an antibody (e.g., a cysteineengineered anti-VEGF antibody) or an antibody conjugate (e.g., amonodisperse HA conjugate) of the invention. For the TAO/PRN regimen,following initial intravitreal injections every four weeks (04W)(typically for about 3 months), the subject is monitored monthly orevery other month (or at even longer intervals), with injectionsadministered in the event of evidence of disease activity (e.g., adecline in visual acuity or fluid on optical coherence tomography(OCT)). For the TAE regimen, a subject may be treated every four weeks(04W), followed by extending the interval of treatment by a fixed numberof weeks (e.g., +2 weeks) for each subsequent visit up to a maximalinterval (e.g., every 6 weeks, ever 8 weeks, every 10 weeks, or every 12weeks). The eye(s) may be observed and treated at each visit, even ifthere is no evidence of disease activity. If the macula appears wet(e.g., by OCT), the interval for injections can be shortened (e.g., −2weeks) until the macula appears dry again. In some instances, the oculardisorder is AMD (e.g., wet AMD).

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or antibody conjugate of the inventioncan occur prior to, simultaneously, and/or following, administration ofthe additional therapeutic agent or agents. In one embodiment,administration of the antibody or antibody conjugate and administrationof an additional therapeutic agent occur within about one, two, three,four, or five months, or within about one, two or three weeks, or withinabout one, two, three, four, five, or six days, of each other.

An antibody (e.g., a cysteine engineered anti-VEGF antibody) or anantibody conjugate (e.g., a monodisperse HA conjugate) of the invention(and any additional therapeutic agent) for prevention or treatment of anocular disease or condition can be administered by any suitable means,including but not limited to, for example, ocular, intraocular, and/orintravitreal injection, and/or juxtascleral injection, and/or subtenoninjection, and/or superchoroidal injection, and/or topicaladministration in the form of eye drops and/or ointment. Such antibodiesor antibody conjugates may be delivered by a variety of methods, forexample, intravitreally as a device and/or a depot that allows for slowrelease of the compound into the vitreous, including those described inreferences such as Intraocular Drug Delivery, Jaffe, Jaffe, Ashton, andPearson, editors, Taylor & Francis (March 2006). In one example, adevice may be in the form of a mini pump and/or a matrix and/or apassive diffusion system and/or encapsulated cells that release thecompound for a prolonged period of time (Intraocular Drug Delivery,Jaffe, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March2006). Additional approaches which may be used are described in SectionG below.

Formulations for ocular, intraocular, or intravitreal administration canbe prepared by methods and using excipients known in the art. Animportant feature for efficient treatment is proper penetration throughthe eye. Unlike diseases of the front of the eye, where drugs can bedelivered topically, retinal diseases typically benefit from a moresite-specific approach. Eye drops and ointments rarely penetrate theback of the eye, and the blood-ocular barrier hinders penetration ofsystemically administered drugs into ocular tissue. Accordingly, amethod of choice for drug delivery to treat retinal disease, such as AMDand CNV, is typically direct intravitreal injection. Intravitrealinjections are usually repeated at intervals which depend on thepatients condition, and the properties and half-life of the drugdelivered. Additional approaches which may be used are described inSection G below.

The amount of antibody or antibody conjugate which will be effective inthe treatment of a particular ocular disorder or condition will dependon the nature of the disorder or condition, and can be determined bystandard clinical techniques. Where possible, it is desirable todetermine the dose-response curve and the pharmaceutical compositions ofthe invention first in vitro, and then in useful animal model systemsprior to testing in humans.

Additional suitable administration means include parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, forexample, by injections, such as intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.Various dosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein. In some instances, an antibodyconjugate of the invention may be administered intravenously,intramuscularly, intradermally, percutaneously, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrapleurally, intratracheally, intrathecally,intranasally, intravaginally, intrarectally, topically, intratumorally,intraperitoneally, peritoneally, intraventricularly, subcutaneously,subconjunctivally, intravesicularly, mucosally, intrapericardially,intraumbilically, intraorbitally, orally, topically, transdermally, byinhalation, by injection, by implantation, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, in cremes, or in lipid compositions

For the prevention or treatment of disease, the appropriate dosage of anantibody (e.g., a cysteine engineered anti-VEGF antibody) or an antibodyconjugate (e.g., a monodisperse HA conjugate) of the invention (whenused alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the type of antibody, the severity and course of the disease, whetherthe antibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody(e.g., a cysteine engineered anti-VEGF antibody) or antibody conjugate(e.g., a monodisperse HA conjugate) is suitably administered to thepatient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1mg/kg, 0.2 mg/kg, 0.4 mg/kg, 0.6 mg/kg, 0.8 mg/kg, 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10mg/kg) of antibody or antibody conjugate can be an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. In someembodiments, the antibody or antibody conjugate used is about 0.01 mg/kgto about 45 mg/kg, about 0.01 mg/kg to about 40 mg/kg, about 0.01 mg/kgto about 35 mg/kg, about 0.01 mg/kg to about 30 mg/kg, about 0.01 mg/kgto about 25 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about 0.01 mg/kgto about 15 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kgto about 5 mg/kg, or about 0.01 mg/kg to about 1 mg/kg. For antibodyconjugates, the dosing may be based on the weight of the antibodycomponent of the conjugate. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs.

In some embodiments, the methods may further comprise an additionaltherapy. The additional therapy may be radiation therapy, surgery,chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,immunotherapy, bone marrow transplantation, nanotherapy, monoclonalantibody therapy, or a combination of the foregoing. The additionaltherapy may be in the form of adjuvant or neoadjuvant therapy. In someembodiments, the additional therapy is the administration of smallmolecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. In some embodiments, the additional therapy isgamma irradiation. In some embodiments, the additional therapy may be aseparate administration of one or more of the therapeutic agentsdescribed above.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-VEGF antibody.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an antibody conjugate of the invention(e.g., any described herein, e.g., in Section G below).

F. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment and/or prevention of the disordersdescribed above is provided. The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, IV solution bags, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is by itself or combined with another compositioneffective for treating, preventing and/or diagnosing the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an antibody composition (e.g., an antibody (e.g., acysteine engineered anti-VEGF antibody) or antibody conjugate thereof)of the invention. The label or package insert indicates that thecomposition is used for treating the condition of choice. Moreover, thearticle of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises anantibody or antibody composition thereof of the invention; andoptionally (b) a second container with a composition contained therein,wherein the composition comprises an additional therapeutic agent. Thearticle of manufacture in this embodiment of the invention may furthercomprise a package insert indicating that the composition(s) can be usedto treat a particular condition. Alternatively, or additionally, thearticle of manufacture may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude any of the antibodies or antibody conjugates thereof describedherein and/or any additional therapeutic agents.

G. Ocular Long-Acting Delivery Approaches

The invention provides compositions for treatment of ocular disorders,which may be used for long-acting delivery of antibodies (e.g.,anti-VEGF antibodies (including any anti-VEGF antibody described herein,such as G6.31 AARR)) to the eye. For example, the invention providesantibody conjugates (e.g., monodisperse HA conjugates) that include ananti-VEGF antibody described herein (e.g., Fab, Fab-C, or cysteineengineered antibody (e.g., ThioFab) conjugates). The invention alsoprovides devices that can be used for ocular administration of anantibody or antibody conjugate described herein. The invention furtherprovides pharmaceutical compositions that include antibodies or antibodyconjugates described herein. These compositions can be used in any ofthe therapeutic methods described herein, for example, methods oftreating an ocular disorder (e.g., AMD (e.g., wet AMD), DME, DR (e.g.,NPDR or PDR), or RVO (e.g., CRVO or BRVO)).

1. Antibody Conjugates

The invention provides antibody conjugates that include an antibody(e.g., an anti-VEGF antibody) and a monodisperse polymer covalentlyattached to the antibody. The antibody (e.g., the anti-VEGF antibody)may be covalently attached to the monodisperse polymer in anirreversible fashion or a reversible fashion. Any suitable monodispersepolymer may be used, including those described herein or others known inthe art.

The invention provides an antibody conjugate that includes an antibodyand a monodisperse polymer (e.g., a monodisperse HA polymer) covalentlyattached to the antibody. The polymer can have a polydispersity index(PDI) of about 1.1 or lower. It is to be understood that the PDI valuecan refer to the PDI value of the polymer used to prepare the antibodyconjugate. For example, in some embodiments, the polymer has a PDIbetween 1.0 to about 1.1 (e.g., between 1 to about 1.1, between 1 toabout 1.09, between 1 to about 1.08, between 1 to about 1.07, between 1to about 1.06, between 1 to about 1.05, between 1 to about 1.04, between1 to about 1.03, between 1 to about 1.02, between 1 to about 1.01,between 1 to about 1.005, between about 1.001 to about 1.1, betweenabout 1.001 to about 1.1, between about 1.001 to about 1.09, betweenabout 1.001 to about 1.08, between about 1.001 to about 1.07, betweenabout 1.001 to about 1.06, between about 1.001 to about 1.05, betweenabout 1.001 to about 1.04, between about 1.001 to about 1.03, betweenabout 1.001 to about 1.02, between about 1.001 to about 1.01, betweenabout 1.001 to about 1.005, between about 1.001 to about 1.004, betweenabout 1.001 to about 1.003, between about 1.001 to about 1.002, betweenabout 1.0001 to about 1.1, between about 1.0001 to about 1.09, betweenabout 1.0001 to about 1.08, between about 1.0001 to about 1.07, betweenabout 1.0001 to about 1.06, between about 1.0001 to about 1.05, betweenabout 1.0001 to about 1.04, between about 1.0001 to about 1.03, betweenabout 1.0001 to about 1.02, between about 1.0001 to about 1.01, betweenabout 1.0001 to about 1.005, between about 1.0001 to about 1.004,between about 1.0001 to about 1.003, between about 1.0001 to about1.002, or between about 1.0001 to about 1.005).

For example, in some embodiments, the monodisperse polymer (e.g.,monodisperse HA polymer) has a PDI of 1.001, about 1.0001, about1.00001, about 1.000001, about 1.0000001, or lower. In some embodiments,the monodisperse polymer (e.g., monodisperse HA polymer) has a PDI of1.0, about 1.001, about 1.002, about 1.003, about 1.004, about 1.005,about 1.006, about 1.007, about 1.008, about 1.009, about 1.01, about1.011, about 1.012, about 1.013, about 1.014, about 1.015, about 1.016,about 1.017, about 1.018, about 1.019, about 1.02, about 1.021, about1.022, about 1.023, about 1.024, about 1.025, about 1.026, about 1.027,about 1.028, about 1.029, about 1.03, about 1.031, about 1.032, about1.033, about 1.034, about 1.035, about 1.036, about 1.037, about 1.038,about 1.039, about 1.04, about 1.041, about 1.042, about 1.043, about1.044, about 1.045, about 1.046, about 1.047, about 1.048, about 1.049,about 1.05, about 1.051, about 1.052, about 1.053, about 1.054, about1.055, about 1.056, about 1.057, about 1.058, about 1.059, about 1.06,about 1.061, about 1.062, about 1.063, about 1.064, about 1.065, about1.066, about 1.067, about 1.068, about 1.069, about 1.07, about 1.071,about 1.072, about 1.073, about 1.074, about 1.075, about 1.076, about1.077, about 1.078, about 1.079, about 1.08, about 1.081, about 1.082,about 1.083, about 1.084, about 1.085, about 1.086, about 1.087, about1.088, about 1.089, about 1.09, about 1.091, about 1.092, about 1.093,about 1.094, about 1.095, about 1.096, about 1.097, about 1.098, about1.099, or about 1.1. In some embodiments, the polymer (e.g., HA polymer)has a PDI of about 1.001.

The monodisperse polymer may be a hydrophilic polymer or a hydrophobicpolymer. It is to be understood that a hydrophilic polymer may be awater-soluble polymer. Any suitable hydrophilic polymer may be used, forexample, a hydrophilic polymer described in International PatentApplication Publication No. WO 2011/066417 and/or Pelegri-O'Day et al.J. Am. Chem. Soc. 136:14323-14332, 2014, which are incorporated hereinby reference in their entirety. Exemplary, non-limiting hydrophilicpolymers that can be used include hyaluronic acid (HA), polyethyleneglycol (PEG; also known as poly(ethylene glycol)) (e.g., straight-chainPEG, branched PEG, comb-like PEG, and dendritic PEG), poly[ethyleneoxide)-co-(methylene ethylene oxide)], poly(poly(ethylene glycol) methylether methacrylate) (pPEGMA), agarose, alginate, carageenans,carboxymethylcellulose, cellulose, cellulose derivatives, chitosan,chondroitin sulfate, collagen, dermatan sulfate, dextran, dextransulfate, fibrin, fibrinogen, fibronectin, fucoidan, gelatin,glycosaminoglycans (GAGs), a glycopolymer, heparin, heparin sulfate, ahighly-branched polysaccharide (e.g., a galactose dendrimer), keratansulfate, methyl cellulose, hydroxypropylmethylcellulose (H PMC),poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), pectins, pectinderivatives, pentosane polysulfate, starch, hydroxylethyl starch (HES),styrene, vitronectin, poly(acrylic acid), poly(methacrylic acid),poly(acrylamide), poly(acrylic acid), poly(amines), poly(amino acids),poly(carboxybetaine) (PCB), polyelectrolytes, poly(glutamic acid) (PGA),poly(glycerol) (PG) (e.g., linear, midfunctional, hyperbranched, orlinear hyperbranched PG), poly(maleic acid), poly(2-oxazoline) (POZ),poly(2-ethyl-2-oxazoline, polysialic acid (PSA), polystyrene,polystyrene derivatives (e.g., charged polystyrene derivatives),poly(styrenesulfonate-co-PEGMA), polyvinylpyrrolidone (PVP),poly(N-acryloylmorpholine) (pNAcM), and copolymers thereof. In someinstances, the polymer is a hydrophobic polymer, for example,poly(lactic-co-glycolic acid) (PLGA), polylactide (PLA), andpolyglycolide (PGA). The polymer may be biodegradable and/orbiocompatible. In particular embodiments, the polymer is HA.

By way of example, the monodisperse polymer (e.g., HA polymer) mayinclude any suitable number of monomers, for example, between 2 andabout 1×10⁴ monomers (e.g., about 10, about 50, about 100, about 200,about 300, about 400, about 500, about 600, about 700, about 800, about900, about 1000, about 2000, about 3000, about 4000, about 5000, about6000, about 7000, about 8000, about 9000, or about 1×10⁴ monomers), ormore. For example, the polymer (e.g., HA polymer) may include betweenabout 50 and about 250 monomers, about 50 and about 500 monomers,between about 50 and about 1000 monomers, between about 50 and about2000 monomers, between about 50 and about 3000 monomers, between about50 and about 4000 monomers, between about 50 and about 5000 monomers,between about 50 and about 6000 monomers, between about 50 and about7000 monomers, between about 50 and about 8000 monomers, between about50 and about 9000 monomers, between about 50 and about 10000 monomers,between about 100 and about 250 monomers, about 100 and about 500monomers, between about 100 and about 1000 monomers, between about 100and about 2000 monomers, between about 100 and about 3000 monomers,between about 100 and about 4000 monomers, between about 100 and about5000 monomers, between about 100 and about 6000 monomers, between about100 and about 7000 monomers, between about 100 and about 8000 monomers,between about 100 and about 9000 monomers, between about 100 and about10000 monomers, between about 250 and about 500 monomers, between about250 and about 1000 monomers, between about 250 and about 2000 monomers,between about 250 and about 3000 monomers, between about 250 and about4000 monomers, between about 250 and about 5000 monomers, between about250 and about 6000 monomers, between about 250 and about 7000 monomers,between about 250 and about 8000 monomers, between about 250 and about9000 monomers, between about 250 and about 10000 monomers. between about500 and about 1000 monomers, between about 500 and about 2000 monomers,between about 500 and about 3000 monomers, between about 500 and about4000 monomers, between about 500 and about 5000 monomers, between about500 and about 6000 monomers, between about 500 and about 7000 monomers,between about 500 and about 8000 monomers, between about 500 and about9000 monomers, or between about 500 and about 10000 monomers. In someinstances, the polymer (e.g., HA polymer) may include about 500monomers.

The invention provides an antibody conjugate that includes an antibody(e.g., an anti-VEGF antibody such as G6.31 AARR) covalently attached toa monodisperse HA polymer. Such antibody conjugates are sometimesreferred to herein as “monodisperse HA conjugates.” The monodisperse HApolymer can have a polydispersity index (PDI) of about 1.1 or lower. Forexample, in some embodiments, the monodisperse HA polymer has a PDIbetween 1.0 to about 1.1 (e.g., between 1 to about 1.1, between 1 toabout 1.09, between 1 to about 1.08, between 1 to about 1.07, between 1to about 1.06, between 1 to about 1.05, between 1 to about 1.04, between1 to about 1.03, between 1 to about 1.02, between 1 to about 1.01,between 1 to about 1.005, between about 1.001 to about 1.1, betweenabout 1.001 to about 1.1, between about 1.001 to about 1.09, betweenabout 1.001 to about 1.08, between about 1.001 to about 1.07, betweenabout 1.001 to about 1.06, between about 1.001 to about 1.05, betweenabout 1.001 to about 1.04, between about 1.001 to about 1.03, betweenabout 1.001 to about 1.02, between about 1.001 to about 1.01, betweenabout 1.001 to about 1.005, between about 1.001 to about 1.004, betweenabout 1.001 to about 1.003, between about 1.001 to about 1.002, betweenabout 1.0001 to about 1.1, between about 1.0001 to about 1.09, betweenabout 1.0001 to about 1.08, between about 1.0001 to about 1.07, betweenabout 1.0001 to about 1.06, between about 1.0001 to about 1.05, betweenabout 1.0001 to about 1.04, between about 1.0001 to about 1.03, betweenabout 1.0001 to about 1.02, between about 1.0001 to about 1.01, betweenabout 1.0001 to about 1.005, between about 1.0001 to about 1.004,between about 1.0001 to about 1.003, between about 1.0001 to about1.002, or between about 1.0001 to about 1.005).

For example, in some embodiments, the monodisperse HA polymer has a PDIof 1.001, about 1.0001, about 1.00001, about 1.000001, about 1.0000001,or lower. In some embodiments, the monodisperse HA polymer has a PDI of1.0, about 1.001, about 1.002, about 1.003, about 1.004, about 1.005,about 1.006, about 1.007, about 1.008, about 1.009, about 1.01, about1.011, about 1.012, about 1.013, about 1.014, about 1.015, about 1.016,about 1.017, about 1.018, about 1.019, about 1.02, about 1.021, about1.022, about 1.023, about 1.024, about 1.025, about 1.026, about 1.027,about 1.028, about 1.029, about 1.03, about 1.031, about 1.032, about1.033, about 1.034, about 1.035, about 1.036, about 1.037, about 1.038,about 1.039, about 1.04, about 1.041, about 1.042, about 1.043, about1.044, about 1.045, about 1.046, about 1.047, about 1.048, about 1.049,about 1.05, about 1.051, about 1.052, about 1.053, about 1.054, about1.055, about 1.056, about 1.057, about 1.058, about 1.059, about 1.06,about 1.061, about 1.062, about 1.063, about 1.064, about 1.065, about1.066, about 1.067, about 1.068, about 1.069, about 1.07, about 1.071,about 1.072, about 1.073, about 1.074, about 1.075, about 1.076, about1.077, about 1.078, about 1.079, about 1.08, about 1.081, about 1.082,about 1.083, about 1.084, about 1.085, about 1.086, about 1.087, about1.088, about 1.089, about 1.09, about 1.091, about 1.092, about 1.093,about 1.094, about 1.095, about 1.096, about 1.097, about 1.098, about1.099, or about 1.1. In some embodiments, the monodisperse HA polymerhas a PDI of about 1.001.

In some instances, the monodisperse HA polymer has a molecular weight ofabout 2.5 megadalton (MDa) or lower (e.g., about 2.5 MDa or lower, about2.4 MDa or lower, about 2.3 MDa or lower, about 2.2. MDa or lower, about2.1 MDa or lower, about 2.0 MDa or lower, about 1.9 MDa or lower, about1.8 MDa or lower, about 1.7 MDa or lower, about 1.6 MDa or lower, about1.5 MDa or lower, about 1.4 MDa or lower, about 1.3 MDa or lower, about1.2 MDa or lower, about 1.1 MDa or lower, about 1.0 MDa or lower, about900 kDa or lower, about 800 kDa or lower, about 700 kDa or lower, about600 kDa or lower, about 500 kDa or lower, about 400 kDa or lower, about300 kDa or lower, about 200 kDa or lower, or about 100 kDa or lower). Insome instances, the HA polymer has a molecular weight of about 1 MDa orlower (e.g., about 1.0 MDa or lower, about 900 kDa or lower, about 800kDa or lower, about 700 kDa or lower, about 600 kDa or lower, about 500kDa or lower, about 400 kDa or lower, about 300 kDa or lower, about 200kDa or lower, or about 100 kDa or lower). In some instances, the HApolymer has a molecular weight between about 25 kDa and about 2.5 MDa(e.g., between about 25 kDa and about 2.5 mDa, between about 25 kDa andabout 2 MDa, between about 25 kDa and about 1.5 MDa, between about 25kDa and about 1 MDa, between about 25 kDa and about 900 kDa, betweenabout 25 kDa and about 800 kDa, between about 25 kDa and about 700 kDa,between about 25 kDa and about 600 kDa, between about 25 kDa and about500 kDa, between about 100 kDa and about 2.5 mDa, between about 100 kDaand about 2 MDa, between about 100 kDa and about 1.5 MDa, between about100 kDa and about 1 MDa, between about 100 kDa and about 900 kDa,between about 100 kDa and about 800 kDa, between about 100 kDa and about700 kDa, between about 100 kDa and about 600 kDa, between about 100 kDaand about 500 kDa, between about 250 kDa and about 2.5 MDa, betweenabout 250 kDa and about 2 MDa, between about 250 kDa and about 1.5 MDa,between about 250 kDa and about 1 MDa, between about 250 kDa and about900 kDa, between about 250 kDa and about 800 kDa, between about 250 kDaand about 700 kDa, between about 250 kDa and about 600 kDa, betweenabout 250 kDa and about 500 kDa, between about 500 kDa and about 2.5MDa, between about 500 kDa and about 2 MDa, between about 500 kDa andabout 1.5 MDa, between about 500 kDa and about 1 MDa, between about 500kDa and about 900 kDa, between about 500 kDa and about 800 kDa, betweenabout 500 kDa and about 700 kDa, between about 500 kDa and about 600kDa, between about 1 MDa and about 2.5 MDa, between about 1 MDa andabout 2 MDa, between about 1 MDa and about 1.5 MDa, between about 1 MDaand about 1.25 MDa, between about 1.25 MDa and about 2.5 MDa, betweenabout 1.25 MDa and about 2 MDa, between about 1.25 MDa and about 1.5MDa, between about 1.5 MDa and about 2.5 MDa, between about 1.5 MDa andabout 2 MDa, between about 1.5 MDa and about 1.75 MDa, or between 1.75MDa and about 2.5 MDa).

In some instances, the monodisperse HA polymer has a molecular weightbetween about 25 kDa and about 500 kDa (e.g., between about 25 kDa andabout 500 kDa, between about 25 kDa and about 450 kDa, between about 25kDa and about 400 kDa, between about 25 kDa and about 350 kDa, betweenabout 25 kDa and about 300 kDa, between about 25 kDa and about 300 kDa,between about 25 kDa and about 250 kDa, between about 25 kDa and about200 kDa, between about 25 kDa and about 150 kDa, between about 25 kDaand about 100 kDa, between about 25 kDa and about 50 kDa, between about40 kDa and about 500 kDa, between about 40 kDa and about 450 kDa,between about 40 kDa and about 400 kDa, between about 40 kDa and about350 kDa, between about 40 kDa and about 300 kDa, between about 40 kDaand about 300 kDa, between about 40 kDa and about 250 kDa, between about40 kDa and about 200 kDa, between about 40 kDa and about 150 kDa,between about 40 kDa and about 100 kDa, between about 40 kDa and about50 kDa, between about 50 kDa and about 500 kDa, between about 50 kDa andabout 450 kDa, between about 50 kDa and about 400 kDa, between about 50kDa and about 350 kDa, between about 50 kDa and about 300 kDa, betweenabout 50 kDa and about 300 kDa, between about 50 kDa and about 250 kDa,between about 50 kDa and about 200 kDa, between about 50 kDa and about150 kDa, between about 50 kDa and about 100 kDa, between about 50 kDaand about 75 kDa, between about 100 kDa and about 500 kDa, between about100 kDa and about 450 kDa, between about 100 kDa and about 400 kDa,between about 100 kDa and about 350 kDa, between about 100 kDa and about300 kDa, between about 100 kDa and about 300 kDa, between about 100 kDaand about 250 kDa, between about 100 kDa and about 200 kDa, betweenabout 100 kDa and about 150 kDa, between about 150 kDa and about 500kDa, between about 150 kDa and about 450 kDa, between about 150 kDa andabout 400 kDa, between about 150 kDa and about 350 kDa, between about150 kDa and about 300 kDa, between about 150 kDa and about 300 kDa,between about 150 kDa and about 250 kDa, between about 150 kDa and about200 kDa, between about 175 kDa and about 500 kDa, between about 175 kDaand about 450 kDa, between about 175 kDa and about 400 kDa, betweenabout 175 kDa and about 350 kDa, between about 175 kDa and about 300kDa, between about 175 kDa and about 300 kDa, between 175 200 kDa andabout 250 kDa, between about 175 kDa and about 225 kDa, between about200 kDa and about 500 kDa, between about 200 kDa and about 450 kDa,between about 200 kDa and about 400 kDa, between about 200 kDa and about350 kDa, between about 200 kDa and about 300 kDa, between about 200 kDaand about 300 kDa, between about 200 kDa and about 250 kDa, or betweenabout 200 kDa and about 225 kDa).

In some instances, the monodisperse HA polymer has a molecular weightbetween about 100 kDa and about 250 kDa (e.g., about 100 kDa, about 110kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, about 200 kDa,about 210 kDa, about 220 kDa, about 230 kDa, about 240 kDa, or about 250kDa). In particular instances, the HA polymer has a molecular weight ofabout 200 kDa.

Any of the preceding molecular weights may be a weight-average molecularweight (also known as weight-average molar mass).

In some instances, any of the preceding monodisperse HA polymers islinear, i.e., not cross-linked.

In other instances, the invention provides an antibody conjugate thatincludes an antibody (e.g., an anti-VEGF antibody such as G6.31 AARR)covalently attached to a monodisperse PEG polymer. Such antibodyconjugates are sometimes referred to as “PEG conjugates” herein. Anysuitable monodisperse PEG polymer may be used. It is to be understoodthat monodisperse PEG polymers may have different PDI values as comparedto monodisperse HA polymers. For example, commercially available PEGpolymers may have a PDI below 1.1; thus, a monodisperse PEG polymerwould be defined by a different range of PDI values compared to amonodisperse HA polymer. For example, a monodisperse PEG polymer mayhave a PDI from about 1 to about 1.02 (e.g., a PDI of 1, about 1.001,about 1.002, about 1.003, about 1.004, about 1.005, about 1.006, about1.007, about 1.008, about 1.009, about 1.01, about 1.011, about 1.012,about 1.013, about 1.014, about 1.015, about 1.016, about 1.017, about1.018, about 1.019, or about 1.02). The PEG may be a branched PEG, astar PEG, or a comb PEG. The PEG polymer may be, for example, a PEGtetramer, a PEG hexamer, or a PEG octamer. In some instances, theantibody conjugate includes an anti-VEGF antibody (e.g., an anti-VEGFantibody described herein, such as G6.31 AARR) covalently attached to aPEG dendrimer. PEG polymers are commercially available, for example,from JenKem Technology, Quanta BioDesign, NOF America Corporation, andother vendors.

In some instances, the monodisperse PEG polymer has a molecular weightbetween about 1 kDa and about 500 kDa (e.g., between about 1 kDa andabout 500 kDa, between about 1 kDa and about 450 kDa, between about 1kDa and about 400 kDa, between about 1 kDa and about 350 kDa, betweenabout 1 kDa and about 300 kDa, between about 1 kDa and about 300 kDa,between about 1 kDa and about 250 kDa, between about 1 kDa and about 200kDa, between about 1 kDa and about 150 kDa, between about 1 kDa andabout 100 kDa, between about 1 kDa and about 50 kDa, between about 10kDa and about 500 kDa, between about 10 kDa and about 450 kDa, betweenabout 10 kDa and about 400 kDa, between about 10 kDa and about 350 kDa,between about 10 kDa and about 300 kDa, between about 10 kDa and about300 kDa, between about 10 kDa and about 250 kDa, between about 10 kDaand about 200 kDa, between about 10 kDa and about 150 kDa, between about10 kDa and about 100 kDa, between about 10 kDa and about 50 kDa, betweenabout 20 kDa and about 500 kDa, between about 20 kDa and about 450 kDa,between about 20 kDa and about 400 kDa, between about 20 kDa and about350 kDa, between about 20 kDa and about 300 kDa, between about 20 kDaand about 300 kDa, between about 20 kDa and about 250 kDa, between about20 kDa and about 200 kDa, between about 20 kDa and about 150 kDa,between about 20 kDa and about 100 kDa, between about 20 kDa and about75 kDa, between about 30 kDa and about 500 kDa, between about 30 kDa andabout 450 kDa, between about 30 kDa and about 400 kDa, between about 30kDa and about 350 kDa, between about 30 kDa and about 300 kDa, betweenabout 30 kDa and about 300 kDa, between about 30 kDa and about 250 kDa,between about 30 kDa and about 200 kDa, between about 30 kDa and about150 kDa, between about 40 kDa and about 500 kDa, between about 40 kDaand about 450 kDa, between about 40 kDa and about 400 kDa, between about40 kDa and about 350 kDa, between about 40 kDa and about 300 kDa,between about 40 kDa and about 300 kDa, between about 40 kDa and about250 kDa, between about 40 kDa and about 200 kDa, between about 50 kDaand about 500 kDa, between about 50 kDa and about 450 kDa, between about50 kDa and about 400 kDa, between about 50 kDa and about 350 kDa,between about 50 kDa and about 300 kDa, between about 50 kDa and about300 kDa, between 50 200 kDa and about 250 kDa, or between about 50 kDaand about 225 kDa).

In some instances, the monodisperse PEG polymer has a molecular weightbetween about 5 kDa and about 250 kDa (e.g., about 1 kDa, about 5 kDa,about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa,about 35 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa,about 80 kDa, about 90 kDa, 100 kDa, about 110 kDa, about 120 kDa, about130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa,about 180 kDa, about 190 kDa, about 200 kDa, about 210 kDa, about 220kDa, about 230 kDa, about 240 kDa, or about 250 kDa). In particularinstances, the PEG polymer has a molecular weight of about 20 kDa. Inother instances, the PEG polymer has a molecular weight of about 40 kDa.

Any of the preceding molecular weights may be a weight-average molecularweight (also known as weight-average molar mass).

In some instances, the monodisperse PEG polymer is a PEG tetramer. PEGtetramers are commercially available, for example, NOF AmericaSUNBRIGHT® PTE-400MA, PTE-200MA, PTE-100MA, and JenKem Technology USA 4arm PEG maleimide (Cat. No. 4ARM-MAL). In some instances, the PEGtetramer has a pentaerythritol core. For example, in some instances, thePEG tetramer includes a structure of formula (I), wherein n isindependently any suitable integer:

In another example, in some instances, the monodisperse PEG polymer is aPEG hexamer. PEG hexamers are commercially available, for example,JenKem Technology USA 6 arm PEG amine (Cat. No. 6ARM(DP)-NH2HCl), or PEGhexamers from Quanta BioDesign. In some instances, the PEG hexamerincludes a dipentylerythritol core.

In some instances, the monodisperse PEG polymer is a PEG octamer. PEGoctamers are commercially available, for example, NOF America SUNBRIGHT®HGEO series or JenKem Technology USA 8 arm PEG maleimide (Cat. No.8ARM(TP)-MAL). In some instances, the PEG octamer may include atripentaerithritol core. For example, in some instances, the PEG octamerincludes a structure of formula (II), wherein n is independently anysuitable integer:

In yet another example, in some instances, the PEG octamer includes atripentaerythritol core.

It is to be understood that any suitable conjugation approach, includingthose described herein and others known in the art, may be used toconjugate an anti-VEGF antibody of the invention to a monodispersepolymer. For example, the monodisperse polymer may be conjugated to anysuitable protein functional group, including a primary amine group, acarboxyl group, a sulfhydryl froup, or a carbonyl group. Any suitablechemical reactive group may be used to target the protein functionalgroup, for example, carbodiimide (e.g., EDC), NHS ester, imidoester,pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetyl(e.g., bromoacetyl or iodoacetyl), pyridyldisulfide, thiosulfonate,vinylsulfone, hydrazine, alkoxyamine, diazirine, aryl azide, isocyanate,or others known in the art. See, for example, Hermanson, BioconjugateTechniques, 3^(rd) Edition, 2013. In particular embodiments, HA (e.g.,monodisperse HA) is modified with maleimide groups (HA-maleimide) andsecond, an antibody that includes a free thiol on a cysteine (e.g.,Fab-C or a cysteine variant (e.g., a THIOMAB™ or ThioFab)) is reactedwith HA-maleimide to form covalent HA-Fab conjugates, for example, asdescribed in Example 1.

Any of the preceding antibody conjugates may have a hydrodynamic radiusbetween about 5 nm and about 200 nm (e.g., about 5 nm, about 10 nm,about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170nm, about 180 nm, about 190 nm, or about 200 nm). In some instances, theantibody conjugate has a hydrodynamic radius between about 5 nm andabout 150 nm (e.g., about 5 nm, about 10 nm, about 20 nm, about 30 nm,about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140nm, or about 150 nm). In some instances, the antibody conjugate has ahydrodynamic radius between about 5 nm and about 100 nm (e.g., about 5nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm,about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm). Insome instances, the antibody conjugate has a hydrodynamic radius betweenabout 5 nm and about 60 nm (e.g., about 5 nm, about 10 nm, about 20 nm,about 30 nm, about 40 nm, about 50 nm, or about 60 nm). In someinstances, the antibody conjugate has a hydrodynamic radius betweenabout 25 nm and about 35 nm (e.g., about 25 nm, about 26 nm, about 27nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm,about 33 nm, about 34 nm, or about 35 nm). In some instances, thehydrodynamic radius is about 28 nm.

In some instances, the antibody conjugate has a hydrodynamic radiusbetween about 10 nm and about 200 nm, between about 10 nm and about 180nm, between about 10 nm and about 160 nm, between about 10 nm and about140 nm, between about 10 nm and about 120 nm, between about 10 nm andabout 100 nm, between about 10 nm and about 80 nm, between about 10 nmand about 60 nm, between about 10 nm and about 50 nm, between about 10nm and about 40 nm, between about 10 nm and about 30 nm, between about20 nm and about 200 nm, between about 20 nm and about 180 nm, betweenabout 20 nm and about 160 nm, between about 20 nm and about 140 nm,between about 20 nm and about 120 nm, between about 20 nm and about 100nm, between about 20 nm and about 80 nm, between about 20 nm and about60 nm, between about 20 nm and about 50 nm, between about 20 nm andabout 40 nm, between about 20 nm and about 30 nm, between about 30 nmand about 200 nm, between about 30 nm and about 180 nm, between about 30nm and about 160 nm, between about 30 nm and about 140 nm, between about30 nm and about 120 nm, between about 30 nm and about 100 nm, betweenabout 30 nm and about 80 nm, between about 30 nm and about 60 nm,between about 30 nm and about 50 nm, between about 30 nm and about 40nm, between about 40 nm and about 200 nm, between about 40 nm and about180 nm, between about 40 nm and about 160 nm, between about 40 nm andabout 140 nm, between about 40 nm and about 120 nm, between about 40 nmand about 100 nm, between about 40 nm and about 80 nm, between about 40nm and about 60 nm, between about 40 nm and about 50 nm, between about50 nm and about 200 nm, between about 50 nm and about 180 nm, betweenabout 50 nm and about 160 nm, between about 50 nm and about 140 nm,between about 50 nm and about 120 nm, between about 50 nm and about 100nm, between about 50 nm and about 80 nm, between about 50 nm and about60 nm, between about 60 nm and about 200 nm, between about 60 nm andabout 180 nm, between about 60 nm and about 160 nm, between about 60 nmand about 140 nm, between about 60 nm and about 120 nm, between about 60nm and about 100 nm, or between about 60 nm and about 80 nm.

In any of the preceding antibody conjugates, the antibody may be anantibody fragment that binds VEGF, for example, an antibody fragment ofan anti-VEGF antibody described herein that binds VEGF. In someinstances, the anti-VEGF antibody is a cysteine engineered anti-VEGFantibody, as described herein (see, e.g., Section 1(8)(d) above). Insome instances, the antibody fragment is selected from the groupconsisting of Fab, Fab′, Fab-C, Fab′-SH, Fv, scFv, and (Fab′)₂fragments. In particular instances, the antibody fragment is an Fab, anFab′, or an Fab-C. In some instances, the antibody fragment is an Fab-C.

Any of the preceding antibody conjugates may have an ocular half-lifethat is increased relative to a reference antibody that is notcovalently attached to the polymer (e.g., the hydrophilic polymer). Insome instances, the ocular half-life is increased at least about 2-fold(e.g., about 2-fold, about 3-fold, about 4-fold, about 5-fold, about6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about12-fold, about 14-fold, about 16-fold, about 18-fold, about 20-fold, ormore) relative to the reference antibody. In some instances, the ocularhalf-life is increased at least about 4-fold relative to the referenceantibody. In some instances, the ocular half-life is a vitrealhalf-life. In some instances, the reference antibody is identical to theantibody of the antibody conjugate. In other cases, the referenceantibody is non-identical to the antibody of the antibody conjugate.

Any of the preceding antibody conjugates may have an ocular clearancethat is that is decreased relative to a reference antibody that is notcovalently attached to the polymer (e.g., the hydrophilic polymer). Insome instances, the clearance is decreased at least about 2-fold (e.g.,about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold,about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold,about 14-fold, about 16-fold, about 18-fold, about 20-fold, or more)relative to the reference antibody. In some instances, the clearance isdecreased at least about 4-fold relative to the reference antibody. Insome instances, the clearance is clearance from the vitreous. In someinstances, the reference antibody is identical to the antibody of theantibody conjugate. In other cases, the reference antibody isnon-identical to the antibody of the antibody conjugate.

In some instances, the time period between two intraocularadministrations (e.g., by intravitreal injection) of any of thepreceding antibody conjugates (e.g., HA conjugates) is at least 1 month,e.g., at least 1 month, at least 5 weeks, at least 6 weeks, at least 7weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, atleast 15 weeks, at least 16 weeks, at least 20 weeks, at least 24 weeks,at least 28 weeks, at least 32 weeks, at least 36 weeks, at least 40weeks, at least 44 weeks, at least 48 weeks, at least 52 weeks or more.In some cases, the maximum period between two intraocularadministrations is no longer then four years, e.g., no longer than threeyears, no longer than two years, or no longer than one year. Theantibody conjugate can be administered, for example, every two to twelvemonths, e.g., every four to ten months. In some instances, the antibodyconjugate is administered every six months.

The invention also provides compositions (e.g., pharmaceuticalcompositions) that include any of the antibody conjugates describedabove. In certain embodiments, the composition comprises one or moreadditional compounds. In certain embodiments, the additional compoundbinds to a second biological molecule selected from the group consistingof IL-1β; IL-6; IL-6R; PDGF; angiopoietin; angiopoietin 2; Tie2; S1P;integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ;erythropoietin; complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2receptor; and proteins genetically linked to age-related maculardegeneration (AMD) risk, such as complement pathway components C2,factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3;HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; andTNFRSF10A. In certain embodiments, the additional compound is anantibody or antigen-binding fragment thereof. For example, in someinstances, the additional compound is a bispecific antibody (e.g., ananti-VEGF/anti-Ang2 bispecific antibody, such as RG-7716 or anybispecific anti-VEGF/anti-Ang2 bispecific antibody disclosed in WO2010/069532 or WO 2016/073157 or a variant thereof. In another example,in some instances, the additional compound is an anti-IL-6 antibody, forexample, EBI-031 (Eleven Biotherapeutics; see, e.g., WO 2016/073890),siltuximab (SYLVANT®), olokizumab, clazakizumab, sirukumab, elsilimomab,gerilimzumab, OPR-003, MEDI-5117, PF-04236921, or a variant thereof. Ina still further example, in some instances, the additional compound isan anti-IL-6R antibody, for example, tocilizumab (ACTEMRA®) (see, e.g.,WO 1992/019579), sarilumab, vobarilizumab (ALX-0061), SA-237, or avariant thereof.

The invention further provides compositions (e.g., pharmaceuticalcompositions) that include any of the antibody conjugates describedabove and an additional VEGF antagonist.

2. Devices

Any of the antibodies (e.g., cysteine engineered anti-VEGF antibodies)or antibody conjugates (e.g., monodisperse HA conjugates) describedherein can be administered to the eye using a port delivery device. Aport delivery device is an implantable, refillable device that canrelease a therapeutic agent (e.g., an anti-VEGF antibody conjugate) overa period of months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or moremonths). Exemplary port delivery devices that may be used include thosefrom ForSight Labs, LLC and/or ForSight VISION4, for example, asdescribed in International Patent Application Publication Nos. WO2010/088548, WO2015/085234, WO 2013/116061, WO 2012/019176, WO2013/040247, and WO 2012/019047, which are incorporated herein byreference in their entirety.

For example, the invention provides port delivery devices that includereservoirs containing any of the antibodies or antibody conjugatesdescribed herein. The port delivery device may further include aproximal region, a tubular body coupled to the proximal region in fluidcommunication with the reservoir, and one or more outlets in fluidcommunication with the reservoir and configured to release thecomposition into the eye. The tubular body may have an outer diameterconfigured to be inserted through an incision or opening in the eye ofabout 0.5 mm or smaller. The device may be about 1 mm to about 15 mm inlength (e.g., about 1 mm, about 2 mm, about 4 mm, about 5 mm, about 6mm, about 7 mm, about 9 mm, about 11 mm, about 13 mm, or about 15 mm inlength). The reservoir may have any suitable volume. In some instances,the reservoir has a volume of about 1 μl to about 100 μl (e.g., about 1μl, about 5 μl, about 10 μl, about 20 μl, about 50 μl, about 75 μl, orabout 100 μl). The device or its constituent parts may be made of anysuitable material, for example, polyimide.

In some instances, the port delivery device includes a reservoircontaining any of the antibodies or antibody conjugates described hereinand one or more additional compounds. In certain embodiments, theadditional compound binds to a second biological molecule selected fromthe group consisting of IL-1β; IL-6; IL-6R; PDGF; angiopoietin;angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin;apelin/APJ; erythropoietin; complement factor D; TNFα; HtrA1; a VEGFreceptor; ST-2 receptor; and proteins genetically linked to AMD risk,such as complement pathway components C2, factor B, factor H, CFHR3,C3b, C5, C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3;TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. In certain embodiments, theadditional compound is an antibody or antigen-binding fragment thereof.For example, in some instances, the additional compound is a bispecificantibody (e.g., an anti-VEGF/anti-Ang2 bispecific antibody, such asRG-7716 or any bispecific anti-VEGF/anti-Ang2 bispecific antibodydisclosed in WO 2010/069532 or WO 2016/073157 or a variant thereof. Inanother example, in some instances, the additional compound is ananti-IL-6 antibody, for example, EBI-031 (Eleven Biotherapeutics; see,e.g., WO 2016/073890), siltuximab (SYLVANT®), olokizumab, clazakizumab,sirukumab, elsilimomab, gerilimzumab, OPR-003, MEDI-5117, PF-04236921,or a variant thereof. In a still further example, in some instances, theadditional compound is an anti-IL-6R antibody, for example, tocilizumab(ACTEMRA®) (see, e.g., WO 1992/019579), sarilumab, vobarilizumab(ALX-0061), SA-237, or a variant thereof.

In some instances, the port delivery device includes any of theantibodies or antibody conjugates described herein and an additionalVEGF antagonist.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1: Linear Hyaluronic Acid (HA) Antibody Conjugates Prepared fromMonodisperse HA for Improved Stability

Conjugation of Fabs to the biopolymer hyaluronic acid (HA) cansignificantly improve retention time of the Fab in the eye, for example,by slowing diffusion of the Fab and, thereby, clearance from thevitreous humor. A two step process has been employed for HA-Fabconjugate production: first, commercial HA is modified with maleimidegroups (HA-maleimide) and second, Fab-C (Fab with a free thiol oncysteine) is reacted with HA-maleimide to form covalent HA-Fabconjugates.

When produced via typical means, HA-protein conjugates can have twoorthogonal components of variability. The first source of variability ispolydispersity, which is contributed by the polydispersity of the HAbackbone (FIG. 1A). The second source is heterogeneity, which iscontributed by differences in the number of Fab molecules attached to agiven HA chain. This second source of variability is dictated by thestochastic nature of maleimide modification of HA chains in the firststep of the HA conjugation process. FIG. 1B shows the results of a MonteCarlo simulation in which each acid group of a 200 kDa HA chain wasgiven a 5% chance of being reacted with a maleimide-containing linker,repeated for 1000 independent HA chains. The results suggest that,although the mean number of maleimides per HA chain across thesimulation was the expected value of 24.7, the absolute range was 11 to42.

It can be desirable to maintain the physical and colloidal stability ofantibody conjugates (e.g., antibody conjugates that include linear HAand the anti-VEGF antibody G6.31 AARR; referred to herein asHA-G6.31.AARR antibody conjugates) in aqueous phase and in vitreoushumor. Based in part on the simulations described above, it isconsidered that HA backbone molecular weight and the Fab loading levelcould be important parameters for physical stability. The Fab loadinglevel refers to the average number of antibody (e.g., G6.31.AARR)molecules attached to each HA chain and is expressed in terms of thepercent of acid groups on the HA backbone that are covalently modifiedwith a Fab moiety (each HA repeating unit contains one modifiable acidgroup on the glucuronic acid saccharide). While other parameters maycontribute to conjugate stability (e.g. specific properties of the Fabincluding net charge, surface charge distribution, hydrophobicity), theyare generally uncontrollable within the confines of this specificmolecule.

To assess the impact of HA MW and Fab loading level on HA-G6.31.AARRphysical stability, conjugates were prepared with three different HAstarting MWs (approximately 40 kDa, 200 kDa and 600 kDa) and varying Fabloading levels, stressed under physiological conditions and monitoredfor physical stability over several months to mimic biological exposure.

(A) Materials and Methods

(i) Materials

Sodium hyaluronate (HA, Lifecore Biomedical, Chaska, Minn.) of threedifferent molecular weights were used in this study. Their properties,as assessed by size exclusion chromatography in-line with refractiveindex and multi-angle light scattering detectors (SEC-RI-MALS), aresummarized in Table 4. Mn indicates number average molecular weight; Mwindicates weight average molecular weight; PDI indicates polydispersityindex; and RH indicates hydrodynamic radius. Table 5 shows data for Mn,Mw, and PDI of various polydisperse HA samples compared to amonodisperse sample as determined by SEC-RI-MALS. The data from Tables 4and 5 are from two different lots of HA-200K, which had differentpolydispersity.

TABLE 4 Properties of HA used in this study Label Name Mn, kDa Mw, kDaPDI R_(H), nm  40K 28.8 45.5 1.581 8.7 200K 143.4 204.3 1.424 23.7 600K481.6 619.8 1.287 35.6

TABLE 5 Properties of polydisperse HA samples compared to a monodispersesample Sample Mn (kDa) Mw (kDa) PDI (Mw/Mn) HA10K 13.7 19.6 1.43 HA20K21.0 32.9 1.57 HA40K 28.7 44.7 1.56 HA100K 66.0 107 1.62 HA200K 116 2041.76 HA350K 206 314 1.53 HA700K 473 657 1.39 Monodisperse HA150K 137.2137.3 1.001

(ii) Synthesis of Maleimide-Functionalized HA (HA-Mal)

HA was modified with maleimide groups using an aqueous reaction with thecoupling reagent4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM) and the linker N-(2-aminoethyl)maleimide trifluoroacetate salt(AEM). HA was dissolved in 100 mM 2-(N-morpholino)ethanesulfonic acid(MES) (pH 5.5) at 2.5 mg/mL and to this solution was added DMTMM and AEMunder stirring. The amounts of DMTMM and AEM added varied and wereselected to target different levels of maleimide functionalizationranging from 2 to 10%. The reaction was heated to 70° C. for 2 hours.

Excess AEM and DMTMM were removed from the reaction via a desaltingprocedure. A HIPREP™ 26/10 Desalting column was mounted on an AKTA™purification system (GE Life Sciences) and equilibrated with 10 mMsodium acetate (pH 4.0) 150 mM NaCl. The reaction was injected neat ontothe column, and the HA-mal peak was collected according to absorbance at302 nm and concentrated to greater than 5 mg/mL using centrifugalultrafiltration devices. The maleimide concentration in the HA-mal stocksolution was measured by absorbance at 302 nm using a UV-visiblespectrophotometer, and the molar ratio of maleimide groups per HA chainwas assessed via size exclusion chromatography with multi-angle lightscattering (SEC-MALS).

(iii) Conjugation of Fab-C to HA-Mal

A solution of Fab-C was pH adjusted to 6.5 using 1 M phosphate (pH 6.5)to a final phosphate concentration of 50 mM andethylenediaminetetraacetic acid (EDTA) was spiked to a finalconcentration of 2.5 mM. The Fab-C solution was stirred and to it wasadded HA-mal diluted into reaction buffer comprised of 10 mM phosphate(pH 6.5), 150 mM NaCl, and 2.5 mM EDTA. The stoichiometry was set at 1.2moles of Fab-C per mole of maleimide in the final reaction, and thevolume was set to give a final protein concentration of 1 mg/mL. Theconjugation reaction was carried out at room temperature under stirring.At 3 hours, mercaptoethanol was added at 2 moles per mole of maleimideto cap unreacted maleimide groups. After 30 minutes the reaction wasdiluted to less than 50 mM NaCl with 10 mM phosphate (pH 6.5).

Purification was carried out using size exclusion chromatography (SEC)to separate free Fab-C and Fab dimer from the conjugate. A HILOAD®26/600 SUPERDEX® 200 μg column (GE Healthcare) was equilibrated with 10mM HisHCl (pH 5.5) 150 mM NaCl and the reaction was injected neat. Peaksassociated with conjugate, Fab dimer, and Fab monomer eluted separatelyand the conjugate peak was collected.

(iv) Analysis of HA-Fab Conjugates by SEC-RI-MALS

Residual free Fab content, total conjugate molar mass, and protein massfraction were assessed by SEC-RI-MALS-QELS (a combination of sizeexclusion chromatography (SEC), refractive index (RI) multi-angle lightscattering (MALS), and quasi-elastic light scattering (QELS)) on anAgilent 1200 HPLC with a Wyatt OPTILAB® T-rEX™ refractive index (RI)detector and Wyatt HELEOS™-II multi-angle light scattering (MALS)detector in-line. For SEC, two columns were run in series: ACCLAIM™7.8×150 mm 1000 Å pore size followed by ACCLAIM™ 7.8×150 mm 300 A poresize with phosphate buffered saline (PBS) (pH 7.4) as the runningbuffer. A bovine serum albumin (BSA) control was used to normalize MALSdetectors and correct for band broadening between detectors. Free Fabcontent was measured by integrating the UV A₂₈₀ peaks corresponding withFab and HA-Fab conjugate. Conjugate molar mass was taken as theweight-average molecular weight (Mw) of the conjugate peak. Protein massfraction was calculated using a protein conjugate analysis using thedifferential refractive index (dRI) and UV A₂₈₀ signals.

(v) HA-G6.31.AARR Conjugate Physiological Stress and Analysis

Purified conjugates were buffer exchanged into PBS and spiked with 2 mMsodium azide and 0.01% polysorbate 20 (PBSTN). The final concentrationswere approximately 5 mg/mL on a Fab basis. The samples were sealed andincubated at 37° C. This condition serves as a surrogate for vitreoushumor (mimicking the pH, temperature, and ionic strength of vitreous).The 5 mg/mL concentration represents a “stressful” concentration forassessing precipitation propensity in vitreous (2 mg dose in 4 mL humanvitreous is equivalent to 0.5 mg/mL Fab). At specified time points,samples were withdrawn, diluted to 1 mg/mL in 10 mM HisHCl (pH 5.5) with0.01% polysorbate 20 and 10% (w/v) trehalose, and assayed for solubleprotein concentration (A₂₈₀), turbidity (A₄₅₀), SEC retention time, andmolecular weight (Mn and Mw by SEC-RI-MALS as above).

(B) Results

(i) Characterization of Materials

Table 6 lists the SEC-RI-MALS characterization results of the nineconjugates used in this study. The conjugates differ in both HA backboneMW (40 kDa, 200 kDa or 600 kDa) and Fab loading level. The sample namesare given as HA backbone MW (kDA) followed by Fab loading level (%).

TABLE 6 SEC-RI-MALS characterization results of HA-G6.31.AARR startingmaterials Sample Name Mn, kDa Mw, kDa Fab loading %  40K 2.8% 146 178.72.78  40K 2.9% 149.4 182.4 2.85  40K 4.7% 191.2 272.4 4.65  40K 6.3%305.9 354.8 6.31 200K 1.3% 442.6 518.7 1.28 200K 2.5% 664.3 813.1 2.45200K 4.7% 1106.2 1362.5 4.66 200K 6.2% 1297.4 1739.3 6.17 600K 2.1%1730.6 2141.3 2.06

The polydispersity of HA-G6.31.AARR conjugates was evaluatedexperimentally using SEC-RI-MALS (FIG. 10). The polydispersity index was1.58, 1.78, and 1.41 for HA40K, HA200K, and HA600K conjugates,respectively.

(ii) Conjugate Stability Under Physiological Stress

Under physiological stress conditions, some HA-G6.31.AARR conjugatesshowed significant physical changes over extended incubation in PBSTN.This change is most clearly evidenced by shifts in the average molecularweight (Mw, weight-average molecular weight) with time, as shown in FIG.2. While some samples did not change in Mw over the 12-week study, thosethat did all showed a decrease in Mw over time. The extent of Mwdecrease was dependent both on the HA backbone molecular weight and theFab loading level. In general, it was observed that precipitationoccurred in samples where the HA molecular weight and Fab loading werehigher.

In order to understand this Mw shift, analysis of the SEC retentionprofiles of incubated HA-G6.31.AARR conjugates was performed. SECretention times shifted to later times upon extended incubation,indicating that the average population of conjugate was becoming smallerprogressively with time (FIG. 3). Because SEC retention time is definedentirely by the backbone HA molecular weight (and not by Fab loading),this observation indicated that either: (a) the entire population ofconjugate molecules was getting smaller over time, or (b) a highermolecular weight subpopulation was precipitating out of solution,resulting in an apparent shift to smaller conjugates. These data arealso summarized in Table 7, in which the differences between retentiontime at study start (T0) and week 12 are shown.

TABLE 7 SEC retention times of HA-G6.31.AARR stability samples at studystart and at 12 weeks after exposure to physiological conditionsRetention Time, Retention Time, Sample T0, min Week 12, min Difference,min  40K 2.8% 13.272 13.267 −0.005  40K 2.9% 13.364 13.272 −0.092  40K4.7% 13.125 13.349 0.224  40K 6.3% 13.292 13.569 0.277 200K 1.3% 11.98611.848 −0.138 200K 2.5% 12.052 12.787 0.735 200K 4.7% 12.31 12.448 0.138200K 6.2% 11.986 12.502 0.516 600K 2.1% 10.692 13.627 2.935

These changes measured by SEC and MALS were paralleled by visualobservations that higher HA backbone molecular weight and higher Fabloading samples contained visible precipitates at later time points.This observation supports the latter explanation for the observed shiftin SEC retention profiles, i.e., that higher molecular weightsubpopulations of HA were precipitating and leading to a shift in theaverage population remaining in solution.

Further supporting this hypothesis is a re-analysis of SEC-RI-MALS dataexamining the distribution of HA-G6.31.AARR conjugates with respect tomolecular weight, as shown in FIG. 4. For the 40K HA backbone sample(left panel), the overall distribution of conjugate molecules withrespect to molecular weight did not change appreciably upon extendedexposure to physiological conditions. For the 600K HA backbone sample(right panel), there was a continuous shift of all population fractionsto smaller molecular weights, indicating that a broad population ofconjugate molecules was precipitating out of solution. For the 200K HAbackbone sample (center panel), the shift in population fractionsoccurred primarily in the higher molecular weight conjugate molecules.This indicates that in the 200K HA backbone sample there was a mixedpopulation of relatively stable and unstable conjugate molecules. Withtime, the higher molecular weight conjugate molecules (relativelyunstable subpopulation) precipitated out of solution, leaving only therelatively stable subpopulation remaining in solution.

(iii) Monodisperse HA Conjugates

Based on the understanding that within the polydisperseHA200K-G6.31.AARR population, only the higher MW subpopulations aredriving the physical instability and precipitation, we hypothesized thatswitching from a polydisperse HA backbone to a monodisperse HA backbonecould result in a more stable antibody conjugate.

Standard commercially-available HA is produced by a multi-step processin which: (a) HA is synthesized by bacterial fermentation resulting inextremely high MW HA (Mw 1-4 MDa), (b) the HA is purified from cellculture, and (c) HA is chemically degraded in a controlled mannerresulting in random scission of HA molecules. This final step of theprocess is critical to the production of lower molecular weight HA thatwould otherwise not be feasible by fermentation alone. However, therandom scission degradation process also results in a broad (i.e.polydisperse) distribution of HA molecular weights.

HA can also be synthesized at commercial scale via a synchronizedchemo-enzymatic process (see Jing et al. J. Biol. Chem. 279:42345-42349,2004; Jing et al. Anal. Biochem. 355:183-188, 2006; and U.S. Pat. No.8,088,604, which are incorporated herein by reference in its entirety).In this process, a purified hyaluronan synthase enzyme is added to amixture of small HA oligosaccharides (typically the HA tetramer, HA4)and the uridine diphosphate (UDP) saccharides UDP-glucuronic acid andUDP-N-acetylglucosamine. The hyaluronan synthase enzyme extends theoligosaccharide fragments using the available sugars in an alternatingmanner resulting in synchronized polymerization of HA. The molecularweight of the resulting HA polymer can be controlled by the ratio of HAsubstrate (e.g. HA4) to sugars in the starting reaction. The result ofthis process is highly monodisperse HA polymers.

For comparison, SEC-RI-MALS characterizations of polydisperse andmonodisperse commercial HA of similar size were compared in FIG. 5. Mostnotable is the difference in polydispersity index, which was 1.779 forpolydisperse HA200K and 1.001 for monodisperse HA150K. This lower levelof polydispersity for monodisperse HA is considered to provide severalkey advantages from the perspective of HA-protein conjugates: (a) easieranalytical characterization because elimination of the HA polydispersityleaves differences in protein loading levels as the only source ofheterogeneity in the sample; (b) elimination of lower and highermolecular weight HA backbones in the overall population, which mayeliminate the instability and precipitation observed in the highermolecular weight HA backbone subpopulations within HA-G6.31.AARRconjugates; and (c) potential reduction of the viscosity of theformulated HA-antibody conjugate.

In the context of using polydisperse and monodisperse HA startingmaterials to prepare G6.31.AARR conjugates, the same difference inpolydispersity was observed: polydisperse HA200K-G6.31.AARR had apolydispersity index of 1.228, while monodisperse HA150K-G6.31.AARR hada polydispersity index of 1.003 (FIG. 6). It is expected that thismonodispersity will improve HA-G6.31.AARR conjugate stability due to theabsence of the higher HA backbone molecular weight subpopulation, whichwas presumed to be responsible for the instability and precipitationobserved under physiological stress of polydisperse HA-G6.31.AARRconjugates.

Example 2: Optimized Fab Loading and Cysteine-Engineered Sites forLinear HA Antibody Conjugates

Covalent conjugation of monoclonal antibodies (mAbs) or antibodyfragments (Fabs) to a polymer scaffold can be performed through a widevariety of chemistries, ranging from amine chemistry (direct amidationthrough solvent-accessible lysine residues) to chemo-enzymaticconjugations using substrate-recognizing enzymes such astransglutaminase. Recently, thiol-maleimide conjugation chemistry hasgained significant interest because it provides several key advantagesover other approaches: (a) it is site-selective, reacting only toreduced, solvent-accessible cysteine residues, (b) it is an extremelyrapid reaction, (c) maleimide-containing linkers are readily availableand are typically easily accessed synthetically, (d) cysteine residuescan typically be easily incorporated into a protein structure, and (e)the thiol-maleimide conjugation can be performed near neutral pH inaqueous conditions.

In order to produce a Fab that is amenable to maleimide conjugation, onestrategy is to produce a Fab-C, in which the heavy chain sequence isextended through the hinge peptide to either the first or second hingedisulfide cysteine position and truncated at that cysteine residue (FIG.7). In practice, this approach can potentially introduce a number ofcomplications that impact conjugation to a polymer scaffold. The primaryconsequence of the spatial proximity of this free cysteine residue tothe interchain disulfide bond is thiol scrambling, in which these threenearby cysteine residues can form three possible disulfideconfigurations. The three possible disulfide configurations areillustrated pictorially in FIGS. 8A-8C. In each configuration, adifferent cysteine residue is reduced, making it the potential site ofconjugation to the maleimide-containing polymer backbone. Theconsequence of this is heterogeneity in the site of attachment betweenthe Fab and the HA backbone, which may have consequences from theperspective of product quality, Fab stability, or safety of theconjugated material.

We hypothesized that relocating the free cysteine residue from theflexible, spatially proximal hinge sequence to a further surfacelocation on the Fab could reduce or eliminate these conjugationvariants. This approach is similar in nature to that employed byTHIOMAB™ cysteine engineered monoclonal antibodies, in which surfaceresidues are mutated to cysteines for later conjugation. In the Fabformat, we term the surface-mutated cysteine-containing Fabs as“ThioFabs.”

Protein-polymer conjugates produced using thiol-maleimide chemistry alsocan suffer from deconjugation of the protein from the polymer backbonethrough a reverse-Michael addition reaction. This can result in slowrelease of free protein from the polymer backbone in a pH- andtemperature-dependent manner. This behavior is also influenced by thelocal chemical environment around the cysteine residue and the structureof the maleimide-containing linker (e.g. presence ofelectron-withdrawing groups or amines). Based on findings onreverse-Michael susceptibility of different cysteine locations inTHIOMAB™ development (see Shen et al. Nat. Biotechnol. 30:184-189,2012), we also predicted that moving the free cysteine from the hingepeptide to a surface location would reduce the rate of reverse-Michaelfree Fab release from a polymer backbone. We also investigated the rateof reverse-Michael deconjugation of a model polyethylene glycol(PEG)-maleimide polymer on Fab-C and ThioFab format molecules underphysiological conditions.

(A) Materials and Methods

(i) Materials

Lys-C enzyme was purchased from Promega (Catalog # V1671, Madison,Wis.), hyaluronidase (recombinant human PH20, also referred to as HAase)enzyme was purchased from Halozyme (San Diego, Calif.), N-ethylmaleimide(NEM) was purchased from Sigma Aldrich (St. Louis, Mo.). Defined methoxypolyethylene glycol maleimide (d-mPEG4-Mal, Part #10745) was purchasedfrom Quanta Biodesign (Plain City, Ohio).

(ii) Limited Lys-C Digestion of G6.31.AARR.Fab-C

A limited Lys-C digest was performed on G6.31.AARR.Fab-C samples.Compared to a more traditional Lys-C digest of a protein, the “limited”Lys-C digest is performed with a reduced quantity of Lys-C enzyme andunder non-denaturing conditions. This results in selective digestion ofthe hinge peptide portion of the G6.31.AARR.Fab-C molecule (KTHTC (SEQID NO: 61)), which is cleaved after the lysine residue. There were foursamples of G6.31.AARR.Fab-C tested with different conditions: (a)+NEM,−Digest, (b)+NEM+Digest, (c) −NEM, +Digest, and (d) −NEM, −Digest.

For each sample, to 500 μg Fab-C in 500 μL of 10 mMhistidine-acetate+150 mM sodium chloride (pH 5.5) was added 50 μl of 1MTris+10 mM NEM (pH 7.5); the NEM was omitted for −NEM samples. Sampleswere incubated for 30 min at 37° C. to cap any free thiols. Lys-C enzymewas then added to +Lys-C samples at a mass ratio of 1:500 Lys-C:Fab-C.Samples were incubated for 30 min at 37° C. After digestion, sampleswere frozen to quench the reaction and analyzed by RP-UPLC-TOF.

(iii) Limited Lys-C Digestion of HA200K-G6.31.AARR

To 500 μg (on a protein basis) of HA200K-G6.31.AARR in 500 μL of 10 mMhistidine-acetate+150 mM sodium chloride buffer (pH 5.5) was added 50 μLof 1M Tris (pH 7.5). Lys-C enzyme was added at a mass ratio of 1:500Lys-C:Fab-C. Samples were incubated overnight at 37° C. After digestion,samples were frozen to quench the reaction and analyzed by RP-UPLC-TOF.

(iv) Hyaluronidase digest of HA200K-G6.31.AARR 500 μg (on a proteinbasis) of HA200K-G6.31.AARR was diluted in 500 μL of 10 mMhistidine-acetate+150 mM sodium chloride (pH 5.5). HAase was added at 10units (U) per 1 μg HA in the conjugate. The reaction mixture wasincubated at 37° C. for 4 h. After digestion, samples were frozen toquench the reaction and analyzed by RP-UPLC-TOF.

(v) Reverse-Michael-mediated deconjugation of model G6.31.AARR-PEGconjugates G6.31.AARR.Fab-C, G6.31.AARR.A140C, G6.31.AARR.L174C, andG6.31.AARR.K149C were buffer exchanged into 10 mM phosphate (pH 6.5) 150mM NaCl 2.5 mM EDTA at between 0.2 and 0.5 mg/mL and d-mPEG4-Mal wasadded at a 20-fold molar excess. The reaction was incubated at roomtemperature for 2 h followed by purification by desalting on PD-10columns (GE Healthcare, Pittsburgh, Pa.) into PBS (pH 7.4). Theconjugates were then concentrated to 1 mg/mL by centrifugalultrafiltration and spiked with oxidized glutathione (GSSG) to a finalconcentration of 2 mM. Constructs were incubated at 37° C. and sampleswere pulled periodically for analysis by RP-UPLC-TOF.

(vi) Reverse-Phase Ultra Performance Liquid Chromatography Time ofFlight (RP-UPLC-TOF) Mass Spectrometry (MS) Analysis

Intact masses of samples were obtained by liquid chromatography-massspectrometry (LC/MS) analysis using an Agilent 6230 electrosprayionization (ESI)-time-of-flight (TOF) mass spectrometer in line with anAgilent 1290 ultraperformance liquid chromatography (UPLC) system.Approximately 2.5 μg of protein was injected per sample and desalted byreverse-phase ultra performance liquid chromatography (RP-UPLC) fordirect online MS analysis. The resulting spectra were deconvoluted tozero-charge state using the MassHunter workstation software/QualitativeAnalysis (Agilent Technologies Inc., Santa Clara, Calif.).

(B) Results

(i) Disulfide Status in G6.31.AARR.Fab-C

To confirm whether the three disulfide states shown in FIGS. 8A-8C existin G6.31.AARR.Fab-C and are maintained in a dynamic thermodynamicequilibrium, we conducted a series of experiments using maleimidecapping using NEM and limited Lys-C digests to probe both theinstantaneous status of the three cysteine residues and their ability tore-arrange dynamically.

In the first experiment, G6.31.AARR.Fab-C was capped with NEM (freezingthe disulfide status due to elimination of any free thiols) and then wassubjected to a limited Lys-C digest, cleaving the heavy chain hingepeptide sequence before the intended free cysteine residue (FIG. 9,second chromatogram). Upon denaturing analysis on RP-UPLC-TOF, speciesassociated with the three disulfide statuses presented in FIGS. 8A-8Cwere observed: (a) intact Fab minus hinge peptide (associated with FIG.8A); (b) light chain plus NEM and cyclized heavy chain (associated withFIG. 8B); and (c) light chain plus hinge peptide and heavy chain minushinge peptide plus NEM (associated with FIG. 8C). This experimentconcretely demonstrated that G6.31.AARR.Fab-C does not exist in ahomogeneous state but contains three distinct species with differentdisulfide configurations and, consequently, reactive cysteine residues.

In the second experiment, G6.31.AARR.Fab-C was subjected to a limitedLys-C digest without NEM capping, leaving the cysteines the possibilityto rearrange dynamically (FIG. 9, third chromatogram). In thisexperiment, nearly all of the RP-UPLC-TOF analyzed protein was in thesame state: Fab minus hinge peptide. Given the findings of the firstexperiment, this second set of data indicates that the three freecysteines rearranged dynamically and on a relatively fast time-scale (30min experimental duration). After cleavage of the hinge peptide byLys-C, the three possible disulfide states “scramble” between the threecysteine residues dynamically. If at some point in time the correctinterchain disulfide is formed, the hinge peptide sequence is left torelease into solution since it was previously cleaved by Lys-C. Theresult is that the sample is driven towards complete correctly-formedinterchain disulfide through this rearrangement process.

These two experiments confirmed that G6.31.AARR.Fab-C can exist in threedistinct states with regard to the three proximal cysteine residues andthat their relative abundances are defined by a dynamic thermodynamicequilibrium. This latter finding is important because it indicates thatno reprocessing step could completely eliminate the two incorrectdisulfide configurations, since given any short period of time the threecysteines would re-scramble to form the three variants observed in thefirst experiment.

(ii) Conjugation Variants in HA-G6.31.AARR.Fab-C Conjugates

While these three disulfide variants exist in G6.31.AARR.Fab-C, itremained to be shown that they also exist as variants in HA-G6.31.AARRconjugates. To elucidate the exact location of conjugation of individualG6.31.AARR.Fab-C molecules to HA-maleimide, we used two enzymaticdigestion procedures. The first, a limited Lys-C digest, cleavedG6.31.AARR at the hinge peptide preceding the intended free heavy chaincysteine residue. In correctly conjugated G6.31.AARR molecules, thistreatment should release free intact G6.31.AARR into solution. However,analysis of digested samples by denaturing RP-UPLC-TOF identified anadditional species in solution: free light chain plus cleaved hingepeptide (FIG. 10A). The presence of this species confirmed that apopulation of G6.31.AARR.Fab-C was conjugated to HA through the heavychain cysteine normally occupied by the interchain disulfide, presumablyoriginating from the disulfide variant depicted in FIG. 8C.

The second enzymatic digestion procedure kept all conjugations intact,but exhaustively digested the HA backbone using hyaluronidase (HAase).This permitted not only analysis of free molecules in solution, but alsoconjugated proteins with small HA oligosaccharides covalently attached.This method permitted direct observation of the conjugated species on anindividual level. In this experiment, G6.31.AARR molecules correctlyconjugated to HA through the intended heavy chain terminal cysteine wereobserved; however, direct evidence of conjugation through the disulfidevariant depicted in FIG. 8B was also found. This was supported by twoobservations: (a) the presence of free cyclized heavy chain molecules insolution; and (b) the presence of HA oligosaccharides with covalentlyattached maleimide linker plus light chain (FIG. 10B). The presence ofthese species confirmed that a population of G6.31.AARR.Fab-C wasconjugated to HA through the light chain cysteine normally occupied bythe interchain disulfide, presumably originating from the disulfidevariant depicted in FIG. 8B.

These two digestion experiments confirmed that disulfide rearrangementdoes lead to variations in the site of attachment betweenG6.31.AARR.Fab-C and HA-maleimide. This observation is expected to applyto any maleimide-containing polymer backbone used for conjugation andany Fab-C containing this configuration of three cysteine residues inclose proximity.

(iii) Designing a Free Cysteine-Containing Fab for More HomogeneousConjugation

The data described above show that three conjugation variants exist whenattaching a Fab-C molecule to a maleimide-containing polymer backbone.This conjugation heterogeneity is caused by the existence of threedistinct disulfide configurations in thermodynamic equilibrium, witheach configuration leaving a different cysteine residue available toaffect conjugation. That these three disulfide configurations arepossible is likely influenced by several factors including the proximityof the intended free hinge cysteine residue to the interchain disulfideand the flexibility of the hinge peptide sequence to which the freecysteine is attached.

To avoid having the free cysteine scramble with the interchaindisulfide, we envisioned moving the free cysteine further from theinterchain disulfide. Therefore, we truncated the hinge sequence astypical for a standard Fab format molecule and mutated surface residuesof the Fab to cysteine residues. The sites were chosen such that theywere sufficiently far from the HVRs so as not to negatively impactantigen binding and were sufficiently far from the interchain disulfideto prevent scrambling.

For an initial screening study, three locations were chosen based onprevious reports from THIOMAB™ development to meet these criteria:LC-K149 (EU numbering), HC-A140 (EU numbering), and HC-L174 (EUnumbering). Each site was mutated to a cysteine residue and the hingepeptide was terminated immediately before the first hinge disulfidecysteine (i.e., KTHT; SEQ ID NO: 87).

To confirm that these cysteine sites were still reactive toHA-maleimide, we performed pilot conjugations under typical conditions(10 mM phosphate (pH 6.5), 150 mM NaCl, and 2.5 mM EDTA) and assessedthe products by SEC-RI-MALS after overnight incubation. Conjugationproceeded normally with generation of HA-G6.31.AARR conjugates at thecorrect retention time compared to a G6.31.AARR.Fab-C conjugationreaction control (FIG. 11). The only major difference noted in theThioFab samples was lower conversion of Fab to conjugate. This is likelycaused by the relatively low pH of the conjugation reaction (pH 6.5),which may be better suited to the pKa of the hinge cysteine compared tothat of the ThioFab engineered cysteine residues. It is expected thatincreasing the pH of the conjugation reaction will improve yields forthe ThioFab conjugations.

(iv) Reverse-Michael Addition Susceptibility of Fab-C and ThioFabFormats

G6.31.AARR in Fab-C and ThioFab formats were conjugated to modelPEG-maleimide constructs and incubated in PBS at 37° C. with GSSG to actas a free thiol trap (i.e., preventing reverse-Michael addition ofreleased free thiol with PEG-maleimide) followed by periodic analysis byRP-UPLC-TOF. In Fab-C-PEG constructs, significant reverse-Michaeldeconjugation was observed over the first seven days, resulting in a9.8% loss of intact conjugate (FIG. 12). ThioFab conjugates showedvariable levels of improvement over Fab-C with the A140C variant showingnearly no deconjugation out to 14 days, the L174C variant showing 5.9%deconjugation, and the K149C variant showing 7.1% deconjugation over thesame time. These data suggest that cysteine mutation variants may beprotective against reverse-Michael-mediated protein deconjugation andmay provide a significant advantage over the Fab-C format in retainingintact protein-polymer conjugates.

Example 3: Exemplary Anti-VEGF Antibodies for Use in the AntibodyConjugates of the Invention

Any of the anti-VEGF antibodies described herein can be used to prepareantibody conjugates as described in Examples 1 and 2. For example, anyanti-VEGF antibody described in International Patent Application No.PCT/US2016/053454 can be used. Table 8 describes exemplary anti-VEGFantibodies that can be used, as well as the amino acid sequences of theVH and VL domains for each antibody. Table 9 describes the VL HVR aminoacid sequences for the anti-VEGF antibodies described in Table 8. Table10 describes the VH HVR amino acid sequences for the anti-VEGFantibodies described in Table 8. In particular embodiments, theanti-VEGF antibody G6.31 AARR (also referred to herein as “G6.31.AARR”)is used.

TABLE 8 VH and VL amino acid sequences for exemplary anti-VEGFantibodies Antibody Name Variant VH (SEQ ID NO) Variant VL (SEQ ID NO)G6.31 WT G6.31 WT (SEQ ID NO: 42) G6.31 WT (SEQ ID NO: 38) LC-N94A G6.31WT (SEQ ID NO: 42) N94A (SEQ ID NO: 41) LC-N94A.LC-F83A G6.31 WT (SEQ IDNO: 42) N94A.F83A (SEQ ID NO: 12) LC-N94A.LC-F83A. A40E.T57E (SEQ ID NO:40) N94A.F83A (SEQ ID NO: 12) HC-A40E.HC-T57E (G6.31 AAEE)N94A.F83A.N82aR.Y58R N82aR.Y58R (SEQ ID NO: 11) N94A.F83A (SEQ ID NO:12) (G6.31 AARR) HCcombo HCcombo (SEQ ID NO: 33) G6.31 WT (SEQ ID NO:38) HCLC2 HCcombo (SEQ ID NO: 33) LCcombo2 (SEQ ID NO: 35) HCLC4 HCcombo(SEQ ID NO: 33) LCcombo4 (SEQ ID NO: 37) HCLC5 HCcombo (SEQ ID NO: 33)N94A.F83A (SEQ ID NO: 12) HCLC3 HCcombo (SEQ ID NO: 33) LCcombo3 (SEQ IDNO: 36) HCLC1 HCcombo (SEQ ID NO: 33) LCcombo1 (SEQ ID NO: 34)R19HCcombo R19HCcombo (SEQ ID NO: 51) G6.31 WT (SEQ ID NO: 38) R19HCLC2R19HCcombo (SEQ ID NO: 51) LCcombo2 (SEQ ID NO: 35) R19HCLC4 R19HCcombo(SEQ ID NO: 51) LCcombo4 (SEQ ID NO: 37) R19HCLC5 R19HCcombo (SEQ ID NO:51) N94A.F83A (SEQ ID NO: 12)

TABLE 9 VL HVR Sequences for Antibodies from Table 8 Antibody NameHVR-L1 HVR-L2 HVR-L3 G6.31 WT RASQDVSTAVA SASFLYS QQGYGNPFT(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 23) LC-N94A RASQDVSTAVASASFLYS QQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10)LC-N94A.LC-F83A RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) LC-N94A.LC-F83A. RASQDVSTAVA SASFLYSQQGYGAPFT HC-A40E.HC-T57E (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10)(G6.31 AAEE) N94A.F83A.N82aR.Y58R RASQDVSTAVA SASFLYS QQGYGAPFT(G6.31 AARR) (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) HCcomboRASQDVSTAVA SASFLYS QQGYGNPFT (SEQ ID NO: 8) (SEQ ID NO: 9)(SEQ ID NO: 23) HCLC2 RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) HCLC4 RASQDVSTAVA SASFLYS QQGYGAPFT(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) HCLC5 RASQDVSTAVA SASFLYSQQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) HCLC3RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9)(SEQ ID NO: 10) HCLC1 RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) R19HCcombo RASQDVSTAVA SASFLYS QQGYGNPFT(SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 23) R19HCLC2 RASQDVSTAVASASFLYS QQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) R19HCLC4RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8) (SEQ ID NO: 9)(SEQ ID NO: 10) R19HCLC5 RASQDVSTAVA SASFLYS QQGYGAPFT (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10)

TABLE 10 VH HVR Sequences for Antibodies from Table 8 Antibody NameHVR-H1 HVR-H2 HVR-H3 G6.31 WT DYWIH GITPAGGYTYYADSVKG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 53) (SEQ ID NO: 3) LC-N94A DYWIHGITPAGGYTYYADSVKG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 53)(SEQ ID NO: 3) LC-N94A.LC-F83A DYWIH GITPAGGYTYYADSVKG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 53) (SEQ ID NO: 3) LC-N94A.LC-F83A. DYWIHGITPAGGYEYYADSVKG FVFFLPYAMDY HC-A40E.HC-T57E (SEQ ID NO: 1)(SEQ ID NO: 21) (SEQ ID NO: 3) (G6.31 AAEE) N94A.F83A.N82aR.Y58R DYWIHGITPAGGYTRYADSVKG FVFFLPYAMDY (G6.31 AARR) (SEQ ID NO: 1) (SEQ ID NO: 7)(SEQ ID NO: 3) HCcombo DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 22) (SEQ ID NO: 3) HCLC2 DYWIHGITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22)(SEQ ID NO: 3) HCLC4 DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1)(SEQ ID NO: 22) (SEQ ID NO: 3) HCLC5 DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 22) (SEQ ID NO: 3) HCLC3 DYWIHGITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22)(SEQ ID NO: 3) HCLC1 DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1)(SEQ ID NO: 22) (SEQ ID NO: 3) R19HCcombo DYWIH GITPAGGYEYYADSVEGFVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22) (SEQ ID NO: 3) R19HCLC2 DYWIHGITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22)(SEQ ID NO: 3) R19HCLC4 DYWIH GITPAGGYEYYADSVEG FVFFLPYAMDY(SEQ ID NO: 1) (SEQ ID NO: 22) (SEQ ID NO: 3) R19HCLC5 DYWIHGITPAGGYEYYADSVEG FVFFLPYAMDY (SEQ ID NO: 1) (SEQ ID NO: 22)(SEQ ID NO: 3)

The upper hinge region of the Fab heavy chain of any of the antibodieslisted above, for example, G6.31 AARR, can be mutated to removereactivity to anti-IgG1 hinge autoantibodies that has been reported inthe literature. See, e.g., Brerski et al., J. Immunol. 181:3183-3192,2008 and Brerski et al., mAbs 2:3, 212-220, 2010. Thus, the C-terminalamino acid of G6.31 AARR heavy chain can be either a T (wild-type (WT)version) or L (variant version that lacks reactivity to anti-human IgGFab). The full-length heavy chain amino acid sequence of wild-type G6.31AARR is SEQ ID NO: 48. The full-length heavy chain amino acid sequenceof the variant version that lacks reactivity to anti-human IgG Fab isSEQ ID NO: 49. The full-length light chain amino acid sequence for bothG6.31 AARR and the variant version that lacks reactivity to anti-humanIgG Fab is SEQ ID NO: 50.

The amino acid sequences of the G6.31.AARR.LC-K149C cysteine engineeredantibody variant light chain and heavy chain are shown below (LC-C149 isin bolded and underlined font).

Light chain (LC): (SEQ ID NO: 89)IQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQQGYGAPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW CVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC.Heavy chain (HC): (SEQ ID NO: 90)EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAGITPAGGYTRYADSVKGRFTISADTSKNTAYLQMRSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT.

The amino acid sequences of the G6.31AARR.HC-A1400 cysteine engineeredantibody variant light chain and heavy chain are shown below (HC-C140 inbold underline font).

LC: (SEQ ID NO: 91) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQQGYGAPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.HC: (SEQ ID NO: 92) EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAGITPAGGYTRYADSVKGRFTISADTSKNTAYLQMRSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT C ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT.

The amino acid sequences of the G6.31AARR.HC-L174C cysteine engineeredantibody variant light chain and heavy chain are shown below (HC-C174 inbold underline font).

LC: (SEQ ID NO: 93) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQQGYGAPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.HC: (SEQ ID NO: 94) EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGLEWVAGITPAGGYTRYADSVKGRFTISADTSKNTAYLQMRSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV C QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT.

Example 4: HA-G6.31.AARR Conjugates Produced from Monodisperse HA ShowImproved Physical Stability Under Physiological Stress Relative toHA-G6.31.AARR Conjugates Produced from Polydisperse HA

(A) Materials and Methods

Monodisperse HA synthesized by chemoenzymatic means using thePasteurella multocida hyaluronan synthase (PmHAS) was from Hyalose(Oklahoma City, Okla.). All other chemicals and reagents were from SigmaAldrich (St. Louis, Mo.).

Monodisperse HA (monoHA, Mw 137 kDa, RH 21.2 nm) was modified withN-(2-aminoethylmaleimide) as previously described to contain 4.0%maleimide groups. G6.31.AARR.Fab-C was then conjugated to themonoHA-maleimide backbone as previously described and purified by SEC ona HILOAD® SUPERDEX® 200 μg column with PBS pH 7.4 running buffer. Theconjugate fractions were pooled and confirmed by SEC-RI-MALS.

Purified monoHA-G6.31.AARR was concentrated by ultrafiltration to 5mg/mL (on a Fab basis) in PBS, spiked with sodium azide and polysorbate20 (PS20) to final concentrations of 2 mM and 0.01%, respectively, andincubated at 37° C. At sampling time points, samples were withdrawn anddiluted to 1 mg/mL in a buffer containing 10 mM His-HCl (pH 5.5), 150 mMNaCl, 10% (w/v) trehalose, and 0.01% polysorbate 20, and then assayedfor soluble protein concentration (A₂₈₀), turbidity (A₄₅₀), SECretention time, and molecular weight (Mn and Mw by SEC-RI-MALS).

(B) Results

HA-G6.31.AARR conjugates produced using monodisperse HA showed vastlyimproved physical stability out to four weeks as compared toHA-G6.31.AARR conjugates produced with similar sized polydisperse HA atthe same Fab loading level (FIG. 14). The molecular weight (Mw) ofmonoHA-G6.31.AARR decreased slightly from week 0 to week 2 and week 4,but this loss could be entirely accounted for by loss in free Fab due toreverse-Michael deconjugation, as commonly observed for thiol-maleimideconjugated proteins. There was also no discernable shift in SECretention time for monoHA-G6.31.AARR, indicating that the physical sizeof the conjugate was not changing over time.

These results clearly support the hypothesis that, within a populationof HA-G6.31.AARR conjugates produced from a polydisperse HA startingmaterial, the high molecular weight HA-based subpopulation isresponsible for the physical instability observed after physiologicalstress. Removing this subpopulation by homogenizing the HA startingmaterial (i.e., starting from a monodisperse HA) results in a morestable conjugate molecule that is less prone to precipitation underphysiological stress conditions. These data further support usingHA-G6.31.AARR conjugates produced from a monodisperse HA material astherapeutic agents for in vivo use.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. An antibody conjugate comprising (i) an antibodyand (ii) a hyaluronic acid (HA) polymer covalently attached to theantibody, wherein the HA polymer has a polydispersity index (PDI) of 1.1or lower.
 2. The antibody conjugate of claim 1, wherein the HA polymerhas a PDI between 1.0 to 1.1.
 3. The antibody conjugate of claim 2,wherein the HA polymer has a PDI between 1.0 to about 1.05.
 4. Theantibody conjugate of any one of claims 1-3, wherein the HA polymer hasa PDI between about 1.0001 to about 1.05.
 5. The antibody conjugate ofclaim 4, wherein the HA polymer has a PDI of about 1.001.
 6. Theantibody conjugate of any one of claims 1-5, wherein the HA polymer hasa molecular weight of about 1 megadalton (MDa) or lower.
 7. The antibodyconjugate of claim 6, wherein the HA polymer has a molecular weightbetween about 25 kDa and about 500 kDa.
 8. The antibody conjugate ofclaim 7, wherein the HA polymer has a molecular weight between about 100kDa and about 250 kDa.
 9. The antibody conjugate of claim 8, wherein theHA polymer has a molecular weight between about 150 kDa and about 200kDa.
 10. The antibody conjugate of any one of claims 1-9, wherein the HApolymer is a linear HA polymer.
 11. The antibody conjugate of any one ofclaims 1-10, wherein the antibody conjugate has a hydrodynamic radiusbetween about 10 nm and about 60 nm.
 12. The antibody conjugate of claim11, wherein the antibody conjugate has a hydrodynamic radius betweenabout 25 nm and about 35 nm.
 13. The antibody conjugate of claim 12,wherein the hydrodynamic radius is about 20 nm to about 30 nm.
 14. Theantibody conjugate of any one of claims 1-13, wherein the antibodyconjugate has an ocular half-life that is increased relative to areference antibody that is not covalently attached to the HA polymer.15. The antibody conjugate of claim 14, wherein the ocular half-life isincreased at least about 2-fold relative to the reference antibody. 16.The antibody conjugate of claim 15, wherein the ocular half-life isincreased at least about 4-fold relative to the reference antibody. 17.The antibody conjugate of any one of claims 14-16, wherein the ocularhalf-life is a vitreal half-life.
 18. The antibody conjugate of any oneof claims 14-17, wherein the reference antibody is identical to theantibody of the antibody conjugate.
 19. The antibody conjugate of anyone of claims 1-18, wherein the antibody specifically binds to abiological molecule selected from the group consisting of vascularendothelial growth factor (VEGF); IL-1β; IL-6; IL-6R; IL-13; IL-13R;PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, andα5β1; betacellulin; apelin/APJ; erythropoietin; complement factor D;TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and a protein geneticallylinked to AMD risk.
 20. The antibody conjugate of claim 19, wherein theVEGF receptor is VEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR.
 21. Theantibody conjugate of claim 19, wherein the protein genetically linkedto AMD risk is selected from the group consisting of complement pathwaycomponents C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1;ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC, COL10A1; andTNFRSF10A.
 22. The antibody conjugate of claim 19, wherein the antibodyspecifically binds to VEGF.
 23. The antibody conjugate of claim 22,wherein the antibody comprises the following six hypervariable regions(HVRs): (a) an HVR-H1 comprising the amino acid sequence of(SEQ ID NO: 1) DYWIH; (b) an HVR-H2 comprising the amino acid sequenceof (SEQ ID NO: 2) GX₁TPX₂GGX₃X₄X₅YX₆DSVX₇X₈, wherein X₁ is Ile orHis, X₂ is Ala or Arg, X₃ is Tyr or Lys, X₄ isThr or Glu, X₅ is Arg, Tyr, Gln, or Glu, X₆ isAla or Glu, X₇ is Lys or Glu, and X₈ is Gly or Glu;(c) an HVR-H3 comprising the amino acid sequence of (SEQ ID NO: 3)FVFFLPYAMDY; (d) an HVR-L1 comprising the amino acid sequence of(SEQ ID NO: 4) RASQX₁VSTAVA, wherein X₁ is Asp or Arg;(e) an HVR-L2 comprising the amino acid sequence of (SEQ ID NO: 5)X₁ASFLYS, wherein X₁ is Ser or Met; and(f) an HVR-L3 comprising the amino acid sequence of (SEQ ID NO: 6)X₁QGYGX₂PFT, wherein X₁ is Gln, Asn, or Thr andX₂ is Ala, Asn, Gln, or Arg.


24. The antibody conjugate of claim 23, wherein the antibody comprisesthe following six HVRs: (a) an HVR-H1 comprising the amino acid sequenceof (SEQ ID NO: 1) DYWIH;(b) an HVR-H2 comprising the amino acid sequence of (SEQ ID NO: 7)GITPAGGYTRYADSVKG, (SEQ ID NO: 21) GITPAGGYEYYADSVKG, or (SEQ ID NO: 22)GITPAGGYEYYADSVEG; (c) an HVR-H3 comprising the amino acid sequence of(SEQ ID NO: 3) FVFFLPYAMDY;(d) an HVR-L1 comprising the amino acid sequence of (SEQ ID NO: 8)RASQDVSTAVA; (e) an HVR-L2 comprising the amino acid sequence of(SEQ ID NO: 9) SASFLYS; and(f) an HVR-L3 comprising the amino acid sequence of (SEQ ID NO: 10)QQGYGAPFT or (SEQ ID NO: 23) QQGYGNPFT.


25. The antibody conjugate of claim 23 or 24, wherein the antibodycomprises the following six HVRs:(a) an HVR-H1 comprising the amino acid sequence of (SEQ ID NO: 1)DYWIH; (b) an HVR-H2 comprising the amino acid sequence of(SEQ ID NO: 7) GITPAGGYTRYADSVKG;(c) an HVR-H3 comprising the amino acid sequence of (SEQ ID NO: 3)FVFFLPYAMDY; (d) an HVR-L1 comprising the amino acid sequence of(SEQ ID NO: 8) RASQDVSTAVA;(e) an HVR-L2 comprising the amino acid sequence of (SEQ ID NO: 9)SASFLYS; and (f) an HVR-L3 comprising the amino acid sequence of(SEQ ID NO: 10) QQGYGAPFT.


26. The antibody conjugate of any one of claims 23-25, wherein theantibody further comprises the following heavy chain variable (VH)domain framework regions (FRs):(a) an FR-H1 comprising the amino acid sequence of (SEQ ID NO: 13)EVQLVESGGGLVQPGGSLRLSCAASGFTIS;(b) an FR-H2 comprising the amino acid sequence of (SEQ ID NO: 14)WVRQAPGKGLEWVA; (c) an FR-H3 comprising the amino acid sequence of(SEQ ID NO: 15) RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR; and(d) an FR-H4 comprising the amino acid sequence of (SEQ ID NO: 16)WGQGTLVTVSS.


27. The antibody conjugate of any one of claims 23-26, wherein theantibody further comprises the following light chain variable (VL)domain FRs: (a) an FR-L1 comprising the amino acid sequence of(SEQ ID NO: 17) DIQMTQSPSSLSASVGDRVTITC;(b) an FR-L2 comprising the amino acid sequence of (SEQ ID NO: 18)WYQQKPGKAPKLLIY; (c) an FR-L3 comprising the amino acid sequence of(SEQ ID NO: 19) GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC; and(d) an FR-L4 comprising the amino acid sequence of (SEQ ID NO: 20)FGQGTKVEIK.


28. The antibody conjugate of claim 23 or 24, wherein the antibodycomprises the following six HVRs:(a) an HVR-H1 comprising the amino acid sequence of (SEQ ID NO: 1)DYWIH; (b) an HVR-H2 comprising the amino acid sequence of(SEQ ID NO: 22) GITPAGGYEYYADSVEG;(c) an HVR-H3 comprising the amino acid sequence of (SEQ ID NO: 3)FVFFLPYAMDY; (d) an HVR-L1 comprising the amino acid sequence of(SEQ ID NO: 8) RASQDVSTAVA;(e) an HVR-L2 comprising the amino acid sequence of (SEQ ID NO: 9)SASFLYS; and (f) an HVR-L3 comprising the amino acid sequence of(SEQ ID NO: 23) QQGYGNPFT.


29. The antibody conjugate of claim 28, wherein the antibody furthercomprises the following VL domain FRs:(a) an FR-L1 comprising the amino acid sequence of (SEQ ID NO: 17) DIQMTQSPSSLSASVGDRVTITC; (b) an FR-L2 comprising the amino acid sequenceof (SEQ ID NO: 18) WYQQKPGKAPKLLIY;(c) an FR-L3 comprising the amino acid sequence of (SEQ ID NO: 24)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC; and(d) an FR-L4 comprising the amino acid sequence of (SEQ ID NO: 20)FGQGTKVEIK.


30. The antibody conjugate of claim 23 or 24, wherein the antibodycomprises the following six HVRs:(a) an HVR-H1 comprising the amino acid sequence of (SEQ ID NO: 1)DYWIH; (b) an HVR-H2 comprising the amino acid sequence of(SEQ ID NO: 22) GITPAGGYEYYADSVEG;(c) an HVR-H3 comprising the amino acid sequence of (SEQ ID NO: 3)FVFFLPYAMDY; (d) an HVR-L1 comprising the amino acid sequence of(SEQ ID NO: 8) RASQDVSTAVA;(e) an HVR-L2 comprising the amino acid sequence of (SEQ ID NO: 9)SASFLYS; and (f) an HVR-L3 comprising the amino acid sequence of(SEQ ID NO: 10) QQGYGAPFT.


31. The antibody of claim 30, wherein the antibody further comprises thefollowing VL domain FRs: (a) an FR-L1 comprising the amino acid sequenceof (SEQ ID NO: 17) DIQMTQSPSSLSASVGDRVTITC, (SEQ ID NO: 25)DIQMTQSPESLSASVGDEVTITC, or (SEQ ID NO: 26) DIQMTQSPSSLSASVGDEVTITC;(b) an FR-L2 comprising the amino acid sequence of (SEQ ID NO: 18)WYQQKPGKAPKLLIY or (SEQ ID NO: 27) WYQQKPGEAPKLLIY;(c) an FR-L3 comprising the amino acid sequence of (SEQ ID NO: 19)GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC or (SEQ ID NO: 28)GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC; and(d) an FR-L4 comprising the amino acid sequence of (SEQ ID NO: 20)FGQGTKVEIK.


32. The antibody of any one of claims 28-31, wherein the antibodyfurther comprises the following VH domain FRs:(a) an FR-H1 comprising the amino acid sequence of (SEQ ID NO: 29)EEQLVEEGGGLVQPGESLELSCAASGFEIS or (SEQ ID NO: 52)EEQLVEEGGGLVQPGESLRLSCAASGFEIS;(b) an FR-H2 comprising the amino acid sequence of (SEQ ID NO: 30)WVRQEPGEGLEWVA; (c) an FR-H3 comprising the amino acid sequence of(SEQ ID NO: 31) RFTISADTSENTAYLQMNELRAEDTAVYYCAR; and(d) an FR-H4 comprising the amino acid sequence of (SEQ ID NO: 32)WGQGELVTVSS.


33. The antibody conjugate of claim 22, wherein the antibody comprises(a) a VH domain comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 11, 40, or42; (b) a VL domain comprising an amino acid sequence having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 12, 41,or 46; or (c) a VH domain as in (a) and a VL domain as in (b).
 34. Theantibody conjugate of claim 33, wherein the VH domain further comprisesthe following FRs: (a) an FR-H1 comprising the amino acid sequence of(SEQ ID NO: 13) EVQLVESGGGLVQPGGSLRLSCAASGFTIS;(b) an FR-H2 comprising the amino acid sequence of (SEQ ID NO: 14)WVRQAPGKGLEWVA or (SEQ ID NO: 39) WVRQEPGKGLEWVA;(c) an FR-H3 comprising the amino acid sequence of (SEQ ID NO: 15)RFTISADTSKNTAYLQMRSLRAEDTAVYYCAR; and(d) an FR-H4 comprising the amino acid sequence of (SEQ ID NO: 16)WGQGTLVTVSS.


35. The antibody conjugate of claim 34, wherein the VH domain comprisesthe amino acid sequence of SEQ ID NO:
 11. 36. The antibody conjugate ofany one of claims 33-35, wherein the VL domain further comprises thefollowing FRs: (a) an FR-L1 comprising the amino acid sequence of(SEQ ID NO: 17) DIQMTQSPSSLSASVGDRVTITC or (SEQ ID NO: 45)DIQMTQSPSSLSASVGDRVTIDC;(b) an FR-L2 comprising the amino acid sequence of (SEQ ID NO: 18)WYQQKPGKAPKLLIY; (c) an FR-L3 comprising the amino acid sequence of(SEQ ID NO: 19) GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC (SEQ ID NO: 44)GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC, or (SEQ ID NO: 54)GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC; and(d) an FR-L4 comprising the amino acid sequence of (SEQ ID NO: 20)FGQGTKVEIK or (SEQ ID NO: 55) FGQGTKVEVK.


37. The antibody conjugate of claim 36, wherein the VL domain comprisesthe amino acid sequence of SEQ ID NO:
 12. 38. The antibody conjugate ofclaim 33, wherein the antibody comprises (a) a VH domain comprising theamino acid sequence of SEQ ID NO: 11 and (b) a VL domain comprising theamino acid sequence of SEQ ID NO:
 12. 39. The antibody conjugate ofclaim 33, wherein the antibody comprises (a) a VH domain comprising theamino acid sequence of SEQ ID NO: 40 and (b) a VL domain comprising theamino acid sequence of SEQ ID NO:
 12. 40. The antibody conjugate ofclaim 33, wherein the antibody comprises (a) a VH domain comprising theamino acid sequence of SEQ ID NO: 42 and (b) a VL domain comprising theamino acid sequence of SEQ ID NO:
 12. 41. The antibody conjugate ofclaim 33, wherein the antibody comprises (a) a VH domain comprising theamino acid sequence of SEQ ID NO: 42 and (b) a VL domain comprising theamino acid sequence of SEQ ID NO:
 41. 42. The antibody conjugate ofclaim 22, wherein the antibody comprises (a) a VH domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 33 or 51; (b) a VL domain comprising anamino acid sequence having at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 12, 34, 35, 36, 37, or 38; or (c) a VHdomain as in (a) and a VL domain as in (b).
 43. The antibody conjugateof claim 42, wherein the antibody further comprises the following FRs:(a) an FR-H1 comprising the amino acid sequence of (SEQ ID NO: 29)EEQLVEEGGGLVQPGESLELSCAASGFEIS or (SEQ ID NO: 52)EEQLVEEGGGLVQPGESLRLSCAASGFEIS;(b) an FR-H2 comprising the amino acid sequence of (SEQ ID NO: 30)WVRQEPGEGLEWVA; (c) an FR-H3 comprising the amino acid sequence of(SEQ ID NO: 31) RFTISADTSENTAYLQMNELRAEDTAVYYCAR; and(d) an FR-H4 comprising the amino acid sequence of (SEQ ID NO: 32)WGQGELVTVSS.


44. The antibody conjugate of claim 43, wherein the VH domain comprisesthe amino acid sequence of SEQ ID NO:
 33. 45. The antibody conjugate ofclaim 43, wherein the VH domain comprises the amino acid sequence of SEQID NO:
 51. 46. The antibody conjugate of any one of claims 42-45,wherein the antibody further comprises the following FRs:(a) an FR-L1 comprising the amino acid sequence of (SEQ ID NO: 17)DIQMTQSPSSLSASVGDRVTITC, (SEQ ID NO: 25) DIQMTQSPESLSASVGDEVTITC, or(SEQ ID NO: 26) DIQMTQSPSSLSASVGDEVTITC;(b) an FR-L2 comprising the amino acid sequence of (SEQ ID NO: 18)WYQQKPGKAPKLLIY or (SEQ ID NO: 27) WYQQKPGEAPKLLIY;(c) an FR-L3 comprising the amino acid sequence of (SEQ ID NO: 19)GVPSRFSGSGSGTDFTLTISSLQPEDAATYYC, (SEQ ID NO: 24)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC, or (SEQ ID NO: 28)GVPSRFSGSGSGTDFTLTIESLQPEDAATYYC; and(d) an FR-L4 comprising the amino acid sequence of (SEQ ID NO: 20)FGQGTKVEIK.


47. The antibody conjugate of claim 46, wherein the VL domain comprisesthe amino acid sequence of SEQ ID NO:
 34. 48. The antibody conjugate ofclaim 46, wherein the VL domain comprises the amino acid sequence of SEQID NO:
 35. 49. The antibody conjugate of claim 46, wherein the VL domaincomprises the amino acid sequence of SEQ ID NO:
 36. 50. The antibodyconjugate of claim 46, wherein the VL domain comprises the amino acidsequence of SEQ ID NO:
 37. 51. The antibody conjugate of claim 46,wherein the VL domain comprises the amino acid sequence of SEQ ID NO:12.
 52. The antibody conjugate of claim 46, wherein the VL domaincomprises the amino acid sequence of SEQ ID NO:
 38. 53. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 38. 54. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 34. 55. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 35. 56. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 36. 57. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 37. 58. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 33 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 12. 59. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 38. 60. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 35. 61. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 37. 62. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a VH domaincomprising the amino acid sequence of SEQ ID NO: 51 and (b) a VL domaincomprising the amino acid sequence of SEQ ID NO:
 12. 63. The antibodyconjugate of claim 42, wherein the antibody comprises (a) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 48 and (b) a lightchain comprising the amino acid sequence of SEQ ID NO:
 50. 64. Theantibody conjugate of claim 42, wherein the antibody comprises (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 49 and (b)a light chain comprising the amino acid sequence of SEQ ID NO:
 50. 65.The antibody conjugate of any one of claims 22-64, wherein the antibodyis capable of inhibiting the binding of VEGF to a VEGF receptor.
 66. Theantibody conjugate of claim 65, wherein the VEGF receptor is VEGFreceptor 1 (Flt-1).
 67. The antibody conjugate of claim 65, wherein theVEGF receptor is VEGF receptor 2 (KDR).
 68. The antibody conjugate ofany one of claims 22-67, wherein the antibody binds human VEGF (hVEGF)with a Kd of about 2 nM or lower.
 69. The antibody conjugate of claim68, wherein the antibody binds hVEGF with a Kd between about 75 pM andabout 2 nM.
 70. The antibody conjugate of claim 69, wherein the antibodybinds hVEGF with a Kd between about 75 pM and about 600 pM.
 71. Theantibody conjugate of claim 70, wherein the antibody binds hVEGF with aKd between about 75 pM and about 500 pM.
 72. The antibody conjugate ofclaim 71, wherein the antibody binds hVEGF with a Kd of about 60 pM. 73.The antibody conjugate of any one of claims 23-27, 33-41, and 63-72,wherein the antibody has a melting temperature (Tm) of greater thanabout 83.5° C.
 74. The antibody conjugate of claim 73, wherein theantibody has a Tm of about 85° C. to about 91° C.
 75. The antibodyconjugate of claim 74, wherein the antibody has a Tm of about 89° C. 76.The antibody conjugate of any one of claims 23, 24, 28-32, 42-62,wherein the antibody has an isoelectric point (pI) of lower than
 8. 77.The antibody conjugate of claim 76, wherein the antibody has a pI offrom about 5 to about
 7. 78. The antibody conjugate of claim 77, whereinthe antibody has a pI of from about 5 to about
 6. 79. The antibodyconjugate of any one of claims 1-78, wherein the antibody is monoclonal,human, humanized, or chimeric.
 80. The antibody conjugate of any one ofclaims 1-79, wherein the antibody is an antigen-binding antibodyfragment.
 81. The antibody conjugate of claim 80, wherein the antibodyfragment is selected from the group consisting of Fab, Fab-C, Fab′-SH,Fv, scFv, and (Fab′)₂ fragments.
 82. The antibody conjugate of claim 81,wherein the antibody fragment is an Fab.
 83. The antibody conjugate ofany one of claims 1-82, wherein the antibody is a monospecific antibody.84. The antibody conjugate of any one of claims 1-82, wherein theantibody is a multispecific antibody.
 85. The antibody conjugate ofclaim 84, wherein the multispecific antibody is a bispecific antibody.86. The antibody conjugate of claim 85, wherein the bispecific antibodybinds VEGF and a second biological molecule selected from the groupconsisting of IL-1β; interleukin-6 (IL-6); interleukin-6 receptor(IL-6R); interleukin-13 (IL-13); IL-13 receptor (IL-13R); PDGF;angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1;betacellulin; apelin/APJ; erythropoietin; complement factor D; TNFα;HtrA1; a VEGF receptor; ST-2 receptor; and a protein genetically linkedto age-related macular degeneration (AMD) risk.
 87. The antibodyconjugate of claim 86, wherein the VEGF receptor is VEGFR1, VEGFR2,VEGFR3, membrane-bound VEGF-receptor (mbVEGFR), or soluble VEGF receptor(sVEGFR).
 88. The antibody conjugate of claim 86, wherein the proteingenetically linked to AMD risk is selected from the group consisting ofcomplement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1;TLR3; TLR4; CETP; LIPC, COL10A1; and TNFRSF10A.
 89. The antibodyconjugate of any one of claims 1-88, wherein the antibody is a cysteineengineered antibody.
 90. The antibody conjugate of claim 89, wherein thecysteine engineered antibody comprises a cysteine mutation in the heavychain selected from the group consisting of HC-A118C, HC-A140C, andHC-L174C (EU numbering), or a cysteine mutation in the light chainselected from the group consisting of LC-K1490 and LC-V205C (Kabatnumbering).
 91. The antibody conjugate of claim 90, wherein the cysteinemutation in the heavy chain is HC-A118C (EU numbering).
 92. The antibodyconjugate of claim 90, wherein the cysteine mutation in the heavy chainis HC-A140C (EU numbering).
 93. The antibody conjugate of claim 90,wherein the cysteine mutation in the heavy chain is HC-L174C (EUnumbering).
 94. The antibody conjugate of claim 90, wherein the cysteinemutation in the light chain is LC-K149C (Kabat numbering).
 95. Theantibody conjugate of claim 90, wherein the cysteine mutation in thelight chain is LC-V205C (Kabat numbering).
 96. The antibody conjugate ofany one of claims 90-95, wherein the HA polymer is covalently attachedto the antibody at the cysteine mutation.
 97. The antibody conjugate ofany one of claims 1-96 for use as a medicament.
 98. The antibodyconjugate of any one of claims 1-96 for use in the manufacture of amedicament for treating an ocular disorder in a subject.
 99. Theantibody conjugate of any one of claims 1-96 for use in reducing orinhibiting angiogenesis in a subject having an ocular disorder.
 100. Theantibody conjugate of any one of claims 1-96 for use in treating anocular disorder in a subject.
 101. The antibody conjugate for use of anyone of claims 98-100, wherein the ocular disorder is selected from thegroup consisting of age-related macular degeneration (AMD), maculardegeneration, macular edema, diabetic macular edema (DME) (includingfocal, non-center DME and diffuse, center-involved DME), retinopathy,diabetic retinopathy (DR) (including proliferative DR (PDR),non-proliferative DR (NPDR), and high-altitude DR), otherischemia-related retinopathies, retinopathy of prematurity (ROP),retinal vein occlusion (RVO) (including central (CRVO) and branched(BRVO) forms), CNV (including myopic CNV), corneal neovascularization, adisease associated with corneal neovascularization, retinalneovascularization, a disease associated with retinal/choroidalneovascularization, pathologic myopia, von Hippel-Lindau disease,histoplasmosis of the eye, familial exudative vitreoretinopathy (FEVR),Coats' disease, Norrie Disease, Osteoporosis-Pseudoglioma Syndrome(OPPG), subconjunctival hemorrhage, rubeosis, ocular neovasculardisease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensiveretinopathy, retinal angiomatous proliferation, macular telangiectasia,iris neovascularization, intraocular neovascularization, retinaldegeneration, cystoid macular edema (CME), vasculitis, papilloedema,retinitis, conjunctivitis (including infectious conjunctivitis andnon-infectious (e.g., allergic) conjunctivitis), Leber congenitalamaurosis, uveitis (including infectious and non-infectious uveitis),choroiditis, ocular histoplasmosis, blepharitis, dry eye, traumatic eyeinjury, and Sjögren's disease.
 102. The antibody conjugate for use ofclaim 101, wherein the ocular disorder is AMD, DME, DR, or RVO.
 103. Theantibody conjugate for use of claim 101 or 102, wherein the oculardisorder is AMD.
 104. The antibody conjugate for use of any one ofclaims 101-103, wherein the AMD is wet AMD.
 105. The antibody conjugatefor use of claim 101 or 102, wherein the ocular disorder is DME.
 106. Apharmaceutical composition comprising the antibody conjugate of any oneof claims 1-96 and a pharmaceutically acceptable carrier, excipient, ordiluent.
 107. The pharmaceutical composition of claim 106, furthercomprising a second agent, wherein the second agent is selected from thegroup consisting of an antibody, an anti-angiogenic agent, a cytokine, acytokine antagonist, a corticosteroid, an analgesic, and a compound thatbinds to a second biological molecule.
 108. The pharmaceuticalcomposition of claim 107, wherein the anti-angiogenic agent is a VEGFantagonist.
 109. The pharmaceutical composition of claim 108, whereinthe VEGF antagonist is an anti-VEGF antibody, an anti-VEGF receptorantibody, a soluble VEGF receptor fusion protein, an aptamer, ananti-VEGF DARPin®, or a VEGFR tyrosine kinase inhibitor.
 110. Thepharmaceutical composition of claim 109, wherein the anti-VEGF antibodyis ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody.111. The pharmaceutical composition of claim 110, wherein the bispecificanti-VEGF antibody is an anti-VEGF/anti-Ang2 antibody.
 112. Thepharmaceutical composition of claim 111, wherein the anti-VEGF/anti-Ang2antibody is RG-7716.
 113. The pharmaceutical composition of claim 109,wherein the soluble VEGF receptor fusion protein is aflibercept(EYLEA®).
 114. The pharmaceutical composition of claim 109, wherein theaptamer is pegaptanib (MACUGEN®).
 115. The pharmaceutical composition ofclaim 109, wherein the anti-VEGF DARPin® is abicipar pegol.
 116. Thepharmaceutical composition of claim 109, wherein the VEGFR tyrosinekinase inhibitor is selected from the group consisting of4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT®(sunitinib).
 117. The pharmaceutical composition of claim 107, whereinthe second biological molecule is selected from the group consisting ofIL-1β; IL-6; IL-6R; IL-13; IL-13R; PDGF; angiopoietin; angiopoietin 2;Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ;erythropoietin; complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2receptor; and a protein genetically linked to AMD risk.
 118. Thepharmaceutical composition of claim 117, wherein the VEGF receptor isVEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR.
 119. The pharmaceuticalcomposition of claim 117, wherein the protein genetically linked to AMDrisk is selected from the group consisting of complement pathwaycomponents C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1;ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC, COL10A1; andTNFRSF10A.
 120. The pharmaceutical composition of any one of claims 107and 117-119, wherein the compound that binds a second biologicalmolecule is an antibody or antigen-binding fragment thereof.
 121. Thepharmaceutical composition of claim 120, wherein the antigen-bindingantibody fragment is selected from the group consisting of Fab, Fab-C,Fab′-SH, Fv, scFv, and (Fab′)₂ fragments.
 122. The pharmaceuticalcomposition of any one of claims 106-121 for use as a medicament. 123.The pharmaceutical composition of any one of claims 106-121 for use inthe manufacture of a medicament for treating an ocular disorder in asubject.
 124. The pharmaceutical composition of any one of claims106-121 for use in reducing or inhibiting angiogenesis in a subjecthaving an ocular disorder.
 125. The pharmaceutical composition of anyone of claims 106-121 for use in treating an ocular disorder in asubject.
 126. The pharmaceutical composition for use of any one ofclaims 123-125, wherein the ocular disorder is selected from the groupconsisting of age-related macular degeneration (AMD), maculardegeneration, macular edema, diabetic macular edema (DME) (includingfocal, non-center DME and diffuse, center-involved DME), retinopathy,diabetic retinopathy (DR) (including proliferative DR (PDR),non-proliferative DR (NPDR), and high-altitude DR), otherischemia-related retinopathies, retinopathy of prematurity (ROP),retinal vein occlusion (RVO) (including central (CRVO) and branched(BRVO) forms), CNV (including myopic CNV), corneal neovascularization, adisease associated with corneal neovascularization, retinalneovascularization, a disease associated with retinal/choroidalneovascularization, pathologic myopia, von Hippel-Lindau disease,histoplasmosis of the eye, familial exudative vitreoretinopathy (FEVR),Coats' disease, Norrie Disease, Osteoporosis-Pseudoglioma Syndrome(OPPG), subconjunctival hemorrhage, rubeosis, ocular neovasculardisease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensiveretinopathy, retinal angiomatous proliferation, macular telangiectasia,iris neovascularization, intraocular neovascularization, retinaldegeneration, cystoid macular edema (CME), vasculitis, papilloedema,retinitis, conjunctivitis (including infectious conjunctivitis andnon-infectious (e.g., allergic) conjunctivitis), Leber congenitalamaurosis, uveitis (including infectious and non-infectious uveitis),choroiditis, ocular histoplasmosis, blepharitis, dry eye, traumatic eyeinjury, and Sjögren's disease.
 127. The pharmaceutical composition foruse of claim 126, wherein the ocular disorder is AMD, DME, DR, or RVO.128. The pharmaceutical composition for use of claim 126 or 127, whereinthe ocular disorder is AMD.
 129. The pharmaceutical composition for useof any one of claims 126-128, wherein the AMD is wet AMD.
 130. Thepharmaceutical composition for use of claim 126 or 127, wherein theocular disorder is DME.
 131. A method of reducing or inhibitingangiogenesis in a subject having an ocular disorder, comprisingadministering to the subject an effective amount of the antibodyconjugate of any one of claims 1-96 or the pharmaceutical composition ofany one of claims 106-121, thereby reducing or inhibiting angiogenesisin the subject.
 132. A method of treating an ocular disorder, the methodcomprising administering an effective amount of the antibody conjugateof any one of claims 1-96 or the pharmaceutical composition of any oneof claims 106-121 to a subject in need of such treatment.
 133. Themethod of claim 131 or 132, wherein the ocular disorder is selected fromthe group consisting of age-related macular degeneration (AMD), maculardegeneration, macular edema, diabetic macular edema (DME) (includingfocal, non-center DME and diffuse, center-involved DME), retinopathy,diabetic retinopathy (DR) (including proliferative DR (PDR),non-proliferative DR (NPDR), and high-altitude DR), otherischemia-related retinopathies, retinopathy of prematurity (ROP),retinal vein occlusion (RVO) (including central (CRVO) and branched(BRVO) forms), CNV (including myopic CNV), corneal neovascularization, adisease associated with corneal neovascularization, retinalneovascularization, a disease associated with retinal/choroidalneovascularization, pathologic myopia, von Hippel-Lindau disease,histoplasmosis of the eye, familial exudative vitreoretinopathy (FEVR),Coats' disease, Norrie Disease, Osteoporosis-Pseudoglioma Syndrome(OPPG), subconjunctival hemorrhage, rubeosis, ocular neovasculardisease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensiveretinopathy, retinal angiomatous proliferation, macular telangiectasia,iris neovascularization, intraocular neovascularization, retinaldegeneration, cystoid macular edema (CME), vasculitis, papilloedema,retinitis, conjunctivitis (including infectious conjunctivitis andnon-infectious (e.g., allergic) conjunctivitis), Leber congenitalamaurosis, uveitis (including infectious and non-infectious uveitis),choroiditis, ocular histoplasmosis, blepharitis, dry eye, traumatic eyeinjury, and Sjögren's disease.
 134. The method of claim 133, wherein theocular disorder is AMD, DME, DR, or RVO.
 135. The method of claim 133 or134, wherein the ocular disorder is AMD.
 136. The method of any one ofclaims 133-135, wherein the AMD is wet AMD.
 137. The method of claim 133or 134, wherein the ocular disorder is DME.
 138. The method of any oneof claims 131-137, further comprising administering to the subject aneffective amount of a second agent, wherein the second agent is selectedfrom the group consisting of an antibody, an anti-angiogenic agent, acytokine, a cytokine antagonist, a corticosteroid, an analgesic, and acompound that binds to a second biological molecule.
 139. The method ofclaim 138, wherein the anti-angiogenic agent is a VEGF antagonist. 140.The method of claim 139, wherein the VEGF antagonist is an anti-VEGFantibody, an anti-VEGF receptor antibody, a soluble VEGF receptor fusionprotein, an aptamer, an anti-VEGF DARPin®, or a VEGFR tyrosine kinaseinhibitor.
 141. The method of claim 140, wherein the anti-VEGF antibodyis ranibizumab (LUCENTIS®), RTH-258, or a bispecific anti-VEGF antibody.142. The method of claim 141, wherein the bispecific anti-VEGF antibodyis an anti-VEGF/anti-Ang2 antibody.
 143. The method of claim 142,wherein the anti-VEGF/anti-Ang2 antibody is RG-7716.
 144. The method ofclaim 140, wherein the soluble VEGF receptor fusion protein isaflibercept (EYLEA®).
 145. The method of claim 140, wherein the aptameris pegaptanib (MACUGEN®).
 146. The method of claim 140, wherein theanti-VEGF DARPin® is abicipar pegol.
 147. The method of claim 140,wherein the VEGFR tyrosine kinase inhibitor is selected from the groupconsisting of4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171), vatalanib (PTK787), semaxaminib (SU5416), and SUTENT®(sunitinib).
 148. The method of claim 138, wherein the second biologicalmolecule is selected from the group consisting of IL-1β; IL-6; IL-6R;IL-13; IL-13R; PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrinsαvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin;complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and aprotein genetically linked to AMD risk.
 149. The method of claim 148,wherein the VEGF receptor is VEGFR1, VEGFR2, VEGFR3, mbVEGFR, or sVEGFR.150. The method of claim 149, wherein the protein genetically linked toAMD risk is selected from the group consisting of complement pathwaycomponents C2, factor B, factor H, CFHR3, C3b, C5, C5a, and C3a; HtrA1;ARMS2; TIMP3; HLA; IL-8; CX3CR1; TLR3; TLR4; CETP; LIPC, COL10A1; andTNFRSF10A.
 151. The method of any one of claims 138 and 148-150, whereinthe compound that binds a second biological molecule is an antibody orantigen-binding fragment thereof.
 152. The method of claim 151, whereinthe antigen-binding antibody fragment is selected from the groupconsisting of Fab, Fab-C, Fab′-SH, Fv, scFv, and (Fab′)₂ fragments. 153.The method of any one of claims 131-152, wherein the antibody conjugateor the pharmaceutical composition is administered intravitreally,ocularly, intraocularly, juxtasclerally, subtenonly, superchoroidally,topically, intravenously, intramuscularly, intradermally,percutaneously, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostatically, intrapleurally,intratracheally, intrathecally, intranasally, intravaginally,intrarectally, topically, intratumorally, intraperitoneally,peritoneally, intraventricularly, subcutaneously, subconjunctivally,intravesicularly, mucosally, intrapericardially, intraumbilically,intraorbitally, orally, transdermally, by inhalation, by injection, byeye drop, by implantation, by infusion, by continuous infusion, bylocalized perfusion bathing target cells directly, by catheter, bylavage, in cremes, or in lipid compositions.
 154. The method of claim153, wherein the antibody conjugate or the pharmaceutical composition isadministered intravitreally, ocularly, intraocularly, juxtasclerally,subtenonly, superchoroidally, or topically.
 155. The method of claim 153or 154, wherein the antibody conjugate or the pharmaceutical compositionis administered intravitreally by injection.
 156. The method of claim153 or 154, wherein the antibody conjugate or the pharmaceuticalcomposition is administered topically by eye drop or ointment.
 157. Themethod of claim 153 or 154, wherein the antibody conjugate or thepharmaceutical composition is administered by a port delivery device.158. The method of any one of claims 131-157, wherein the subject is ahuman.